Human desaturase gene and uses thereof

ABSTRACT

The subject invention relates to the identification of a gene involved in the desaturation of polyunsaturated fatty acids at carbon 5 (i.e., “human Δ5-desaturase”) and to uses thereof. In particular, human Δ5-desaturase may be utilized, for example, in the conversion of dihomo-γ-linolenic acid (DGLA) to arachidonic acid (AA) and in the conversion of 20:4n-3 to eicosapentaenoic acid (EPA). AA or polyunsaturated fatty acids produced therefrom may be added to pharmaceutical compositions, nutritional compositions, animal feeds, as well as other products such as cosmetics.

The subject application is a division of allowed U.S. patent applicationSer. No. 09/227,613 filed on Jan. 8, 1999, now U.S. Pat. No. 6,432,684,which is a Continuation-In-Part of pending International ApplicationPCT/US98/07422 filed on Apr. 10, 1998 (which designates the U.S.) whichis a Continuation-In-Part of U.S. Ser. No. 08/833,610, filed Apr. 11,1997, now U.S. Pat. No. 5,972,664, all of which are hereby incorporatedin their entirety by reference.

BACKGROUND OF THE INVENTION

1. Technical Field

The subject invention relates to the identification and isolation of agene that encodes an enzyme (i.e., human Δ5-desaturase) involved in thesynthesis of polyunsaturated fatty acids and to uses thereof. Inparticular, Δ5-desaturase catalyzes the conversion of, for example,dihomo-γ-linolenic acid (DGLA) to arachidonic acid (AA) and(n-3)-eicosatetraenoic acid (20:4n-3) to eicosapentaenoic acid(20:5n-3). The converted product may then be utilized as a substrate inthe production of other polyunsaturated fatty acids (PUFAs). The productor other polyunsaturated fatty acids may be added to pharmaceuticalcompositions, nutritional composition, animal feeds as well as otherproducts such as cosmetics.

2. Background Information

Desaturases are critical in the production of long-chain polyunsaturatedfatty acids which have many important functions. For example, PUFAs areimportant components of the plasma membrane of a cell, where they arefound in the form of phospholipids. They also serve as precursors tomammalian prostacyclins, eicosanoids, leukotrienes and prostaglandins.Additionally, PUFAs are necessary for the proper development of thedeveloping infant brain as well as for tissue formation and repair. Inview of the biological significance of PUFAs, attempts are being made toproduce them, as well as intermediates leading to their production, inan efficient manner.

A number of enzymes are involved in PUFA biosynthesis includingΔ5-desaturase (see FIG. 11). For example, elongase (elo) catalyzes theconversion of γ-linolenic acid (GLA) to dihomo-γ-linolenic acid (DGLA)and of stearidonic acid (18:4n-3) to (n-3)-eicosatetraenoic acid(20:4n-3). Linoleic acid (LA, 18:2-Δ9, 12 or 18:2n-6) is produced fromoleic acid (18:1-Δ9) by a Δ12-desaturase. GLA (18:3-Δ6, 9, 12) isproduced from linoleic acid by a Δ6-desaturase.

It must be noted that animals cannot desaturate beyond the Δ9 positionand therefore cannot convert oleic acid into linoleic acid. Likewise,α-linolenic acid (ALA, 18:3-Δ9, 12, 15) cannot be synthesized bymammals. However, α-linolenic acid can be converted to stearidonic acid(STA, 18:4-Δ6, 9, 12, 15) by a Δ6-desaturase (see PCT publication WO96/13591 and The Faseb Journal, Abstracts, Part I, Abstract 3093, pageA532 (Experimental Biology 98, San Francisco, Calif., Apr. 18-22, 1998)see also U.S. Pat. No. 5,552,306), followed by elongation to(n-3)-eicosatetraenoic acid (20:4−Δ8, 11, 14, 17) in mammals and algae.This polyunsaturated fatty acid (i.e., 20:4-Δ8, 11, 14, 17) can then beconverted to eicosapentaenoic acid (EPA, 20:5-Δ5, 8, 11, 14, 17) by aΔ5-desaturase, such as that of the present invention. Other eukaryotes,including fungi and plants, have enzymes which desaturate at carbon 12(see PCT publication WO 94/11516 and U.S. Pat. No. 5,443,974) and carbon15 (see PCT publication WO 93/11245). The major polyunsaturated fattyacid of animals therefore are either derived from diet and/or fromdesaturation and elongation of linoleic acid or α-linolenic acid. Inview of these difficulties, it is of significant interest to isolategenes involved in PUFA synthesis from species that naturally producethese fatty acids and to express these genes in a microbial, plant, oranimal system which can be altered to provide production of commercialquantities of one or more PUFAs. One of the most important long chainPUFAs, noted above, is arachidonic acid (AA). AA is found in filamentousfungi and can also be purified from mammalian tissues including theliver and adrenal glands. As noted above, AA production fromdihomo-γ-linolenic acid is catalyzed by a Δ5-desaturase. EPA is anotherimportant long-chain PUFA. EPA is found in fungi and also in marineoils. As noted above, EPA is produced from (n-3)-eicosatetraenoic acidand is catalyzed by a Δ5-desaturase.

In view of the above discussion, there is a definite need for theΔ5-desaturase enzyme, the gene encoding this enzyme, as well asrecombinant methods of producing this enzyme. Additionally, a needexists for oils containing levels of PUFAs beyond those naturallypresent as well as those enriched in novel PUFAs. Such oils can only bemade by isolation and expression of the Δ5-desaturase gene.

All U.S. patents and publications referred to herein are herebyincorporated in their entirety by reference.

SUMMARY OF THE INVENTION

The present invention includes an isolated nucleotide sequencecorresponding to or complementary to at least about 50% of thenucleotide sequence shown in SEQ ID NO:1 (FIG. 12).

The isolated nucleotide sequence may be represented by SEQ ID NO:1.These sequences may encode a functionally active desaturase whichutilizes a polyunsaturated fatty acid as a substrate. The sequences maybe derived from a mammal such as, for example, a human.

The present invention also includes purified proteins encoded by thenucleotide sequences referred to above. Additionally, the presentinvention includes a purified polypeptide which desaturatespolyunsaturated fatty acids at carbon 5 and has at least about 50% aminoacid similarity to the amino acid sequence of the purified proteinsreferred to directly above.

Furthermore, the present invention also encompasses a method ofproducing a human Δ5-desaturase. This method comprises the steps of: a)isolating the nucleotide sequence represented by SEQ ID NO:1 (FIG. 12);b) constructing a vector comprising: i) the isolated nucleotide sequenceoperably linked to ii) a promoter; and c) introducing the vector into ahost cell under time and conditions sufficient for expression of thehuman Δ5-desaturase. The host cell may be, for example, a eukaryoticcell or a prokaryotic cell. In particular, the prokaryotic cell may be,for example, E. coli, cyanobacteria or B. subtilis. The eukaryotic cellmay be, for example, a mammalian cell, an insect cell, a plant cell or afungal cell (e.g., a yeast cell such as Saccharomyces cerevisiae,Saccharomyces carlsbergensis, Candida spp., Lipomyces starkey, Yarrowialipolytica, Kluyveromyces spp., Hansenula spp., Trichoderma spp. orPichia spp.).

Additionally, the present invention also encompasses a vectorcomprising: a) a nucleotide sequence as represented by SEQ ID NO:1 (FIG.12) operably linked to b) a promoter. The invention also includes a hostcell comprising this vector.

The host cell may be, for example, a eukaryotic cell or a prokaryoticcell. Suitable eukaryotic cells and prokaryotic cells are as definedabove.

Moreover, the present invention also includes a plant cell, plant orplant tissue comprising the above vector, wherein expression of thenucleotide sequence of the vector results in production of apolyunsaturated fatty acid by the plant cell, plant or plant tissue. Thepolyunsaturated fatty acid may be, for example, selected from the groupconsisting of AA and EPA. The invention also includes one or more plantoils or acids expressed by the above plant cell, plant or plant tissue.

Additionally, the present invention also encompasses a transgenic plantcomprising the above vector, wherein expression of the nucleotidesequence of the vector results in production of a polyunsaturated fattyacid in seeds of the transgenic plant.

Also, the invention includes a mammalian cell comprising the abovevector wherein expression of the nucleotide sequence of the vectorresults in production of altered levels of AA or EPA when the cell isgrown in a culture media comprising a fatty acid selected from the groupconsisting of an essential fatty acid, LA and ALA.

It should also be noted that the present invention encompasses atransgenic, non-human mammal whose genome comprises a DNA sequenceencoding a human Δ5-desaturase operably linked to a promoter. The DNAsequence may be represented by SEQ ID NO:1 (FIG. 12). Additionally, thepresent invention includes a fluid (e.g., milk) produced by thetransgenic, non-human mammal wherein the fluid comprises a detectablelevel of at least human Δ5-desaturase.

Additionally, the present invention includes a method (i.e., “first”method) for producing a polyunsaturated fatty acid comprising the stepsof: a) isolating the nucleotide sequence represented by SEQ ID NO:1(FIG. 12); b) constructing a vector comprising the isolated nucleotidesequence; c) introducing the vector into a host cell under time andconditions sufficient for expression of the human Δ5-desaturase enzyme;and d) exposing the expressed human Δ5-desaturase enzyme to a substratepolyunsaturated fatty acid in order to convert the substrate to aproduct polyunsaturated fatty acid. The substrate polyunsaturated fattyacid may be, for example, DGLA or 20:4n-3 and the productpolyunsaturated fatty acid may be, for example, AA or EPA, respectively.This method may further comprise the step of exposing the productpolyunsaturated fatty acid to an elongase in order to convert theproduct polyunsaturated fatty acid to another polyunsaturated fatty acid(i.e., “second” method). In this method containing the additional step(i.e., “second” method), the product polyunsaturated fatty acid may be,for example, AA or EPA, and the “another” polyunsaturated fatty acid maybe adrenic acid or (n-3)-docosapentaenoic acid, respectively. The methodcontaining the additional step may further comprise a step of exposingthe another polyunsaturated fatty acid to an additional desaturase inorder to convert the another polyunsaturated fatty acid to a finalpolyunsaturated fatty acid (i.e., “third” method). The finalpolyunsaturated fatty acid may be, for example, (n-6)-docosapentaenoicacid or docosahexaenoic (DHA) acid.

The present invention also encompasses a nutritional compositioncomprising at least one polyunsaturated fatty acid selected from thegroup consisting of the product polyunsaturated fatty acid producedaccording to the “first” method, another polyunsaturated fatty acidproduced according to the “second” method, and the final polyunsaturatedfatty acid produced according to the “third” method. The productpolyunsaturated fatty acid may be, for example, AA or EPA. The anotherpolyunsaturated fatty acid may be, for example, adrenic acid or(n-3)-docosapentaenoic acid. The final polyunsaturated fatty acid maybe, for example, (n-6)-docosapentaenoic acid or DHA. This nutritionalcomposition, may be, for example, an infant formula, a dietarysupplement or a dietary substitute and may be administered to a human orto an animal. It may be administered enterally or parenterally. Thenutritional composition may further comprise at least one macronutrientselected from the group consisting of coconut oil, soy oil, canola oil,monoglycerides, diglycerides, glucose, edible lactose, electrodialysedwhey, electrodialysed skim milk, milk whey, soy protein, and proteinhydrolysates. Additionally, the composition may further comprise atleast one vitamin selected from the group consisting of Vitamins A, C,D, E, and B complex and at least one mineral selected from the groupconsisting of calcium magnesium, zinc, manganese, sodium, potassium,phosphorus, copper, chloride, iodine, selenium and iron.

Furthermore, the present invention also includes a a pharmaceuticalcomposition comprising 1) at least one polyunsaturated fatty acidselected from the group consisting of the product polyunsaturated fattyacid produced according to the “first” method, the anotherpolyunsaturated fatty acid produced according to the “second” method,and the final polyunsaturated fatty acid produced according to the“third” method and 2) a pharmaceutically acceptable carrier. Again, thepharmaceutical composition may be administered to a human or to ananimal.

The composition may further comprise an element selected from the groupconsisting of a vitamin, a mineral, a carbohydrate, an amino acid, afree fatty acid, a phospholipid, an antioxidant, and a phenoliccompound.

Additionally, the present invention includes an animal feed comprisingat least one polyunsaturated fatty acid selected from the groupconsisting of the product polyunsaturated fatty acid produced accordingto the first method, the another polyunsaturated fatty acid producedaccording to the second method and the final polyunsaturated fatty acidproduced according to the third method. The product polyunsaturatedfatty acid may be, for example, AA or EPA. The another polyunsaturatedfatty acid may be, for example, adrenic acid or (n-3)-docosapentaenoicacid. The final polyunsaturated fatty acid may be, forexample,(n-6)-docosapentaenoic acid or DHA.

Moreover, the present invention also includes a cosmetic comprising apolyunsaturated fatty acid selected from the group consisting of theproduct polyunsaturated fatty acid produced according to the firstmethod, the another polyunsaturated fatty acid produced according to thesecond method, and the final polyunsaturated fatty acid producedaccording to the third method.

Additionally, the present invention encompasses a method of preventingor treating a condition caused by insufficient intake of polyunsaturatedfatty acids comprising administering to the patient the nutritionalcomposition of above in an amount sufficient to effect prevention ortreatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 outlines the sections of the M. alpina Δ5- and Δ6-desaturases,the clone ID's from the LifeSeq database to which those sections hadhomology, and the keyword associated with the clone ID's.

FIG. 2 represents the contig 2692004.

FIG. 3 represents the contig 2153526.

FIG. 4 represents the contig 3506132.

FIG. 5 represents the contig 3854933.

FIG. 6 represents the contig 2511785.

FIG. 7 represents the contig 2535 generated based on contig 2511785 ofFIG. 6 and contig 3506132 of FIG. 4.

FIG. 8 represents the contig 253538a generated based on contig 2535 ofFIG. 7 and contig 3854933 of FIG. 5.

FIG. 9 represents the amino acid sequence identity between the M. alpinaΔ5-desaturase (Ma29) and the contig 253538a.

FIG. 10 represents the amino acid sequence identity between the M.alpina Δ6-desaturase (Ma524) and the contig 253538a.

FIG. 11 represents various fatty acid biosynthesis pathways. The role ofthe Δ5-desaturase enzyme should be noted.

FIG. 12 represents the complete nucleotide sequence of the humanΔ5-desaturase gene (human Δ5).

FIG. 13 represents the amino acid sequence of the human Δ5-desaturasetranslated from human Δ5 (see FIG. 12).

FIG. 14 illustrates the sequence identity between the pRAE-7 and pRAE-8clones.

FIG. 15 represents the complete putative human desaturase gene sequencefrom clone pRAE-7.

FIG. 16 illustrates the amino acid sequence identity between theputative human desaturase gene in pRAE-7 and the M. alpinaΔ5-desaturase.

FIG. 17 illustrates the amino acid sequence identity between theputative human desaturase gene in pRAE-7 and the M. alpinaΔ6-desaturase.

FIG. 18 illustrates the amino acid sequence identity between theputative human desaturase gene in pRAE-7 and the contig 2535.

FIG. 19 illustrates the amino acid sequence identity between theputative human desaturase gene in pRAE-7 and the contig 38.

FIG. 20 illustrates the amino acid sequence identity between theN-terminus of clone A-1, a representative of Group 1, and the N-terminusof cytochrome b5 gene.

FIG. 21 illustrates the nucleotide sequence identity between thenucleotide sequence of a portion of clone A-1 and a portion of theGenBank sequence ac004228.

FIG. 22 represents the nucleotide sequence identity between thenucleotide sequence of a portion of clone 3-5 of Group 2 and a portionof the GenBank sequence ac004228. Clone 3-5 has an ATG within a NcoIsite, but translates four stops between the ATG and the BamHI site.

FIG. 23 represents the nucleotide sequence identity between thenucleotide sequence of a portion of clone A-10 of Group 3 and a portionof the GenBank sequence ac004228. Clone A-10 has an ATG 135 bp upstreamof the BamHI site, giving an open reading frame of 1267 bp.

FIG. 24 represents the nucleotide sequence identity between thenucleotide sequence of a portion of clone A-16 of Group 4 and a portionof the GenBank sequence ac004228. Clone A-16 does not have an ATG;however, there is an ATG (underlined) upstream of where the sequencealigns with ac004228.

FIG. 25 represents the nucleotide sequence identity between thenucleotide sequence of a portion of clone A-19 of Group 5 and a portionof the GenBank sequence ac004228. Clone A-19 does not have an ATG;however, this clone matches the ac004228 sequence even upstream of theBamHI site.

FIG. 26 represents the partial nucleotide sequence of the GenBanksequence ac004228 and the representative clones from the five Groups.

FIG. 27 represents the nucleotide sequence identity between the humanΔ5-desaturase and contig 3381584.

FIG. 28 represents the nucleotide sequence identity between the humanΔ5-desaturase and contig 2153526.

FIG. 29 represents the amino acid sequence identity between the humanΔ5-desaturase and contig 253538a.

FIG. 30 represents the amino acid sequence identity between the humanΔ5-desaturase and contig 38.

FIG. 31 represents the amino acid sequence identity between the M.alpina Δ6-desaturase (Ma524) and the human the Δ5-desaturase.

FIG. 32 represents the amino acid sequence identity between the M.alpina Δ5-desaturase (Ma29) and the human Δ5-desaturase.

FIG. 33 illustrates the human Δ5-desaturase activity of the gene inclone pRAE-28-5, compared to that in pRAE-26-1, pRAE-33, and pRAE-35,when expressed in baker's yeast.

FIG. 34 illustrates the substrate specificity of the human Δ5-desaturasegene in clone pRAE-28-5, converting DGLA(20:3n-6) to AA(20:4n-6), whenthe gene is expressed in baker's yeast.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention relates to the nucleotide and amino acid sequenceof the Δ5-desaturase gene derived from humans. Furthermore, the subjectinvention also includes uses of the gene and of the enzyme encoded bythis gene. For example, the gene and corresponding enzyme may be used inthe production of polyunsaturated fatty acids such as, for instance,arachidonic acid, eicosapentaenoic acid, and/or adrenic acid which maybe added to pharmaceutical compositions, nutritional compositions and toother valuable products.

The Human Δ5-Desaturase Gene and Enzyme Encoded Thereby

As noted above, the enzyme encoded by the human Δ5-desaturase gene isessential in the production of highly unsaturated polyunsaturated fattyacids having a length greater than 20 carbons. The nucleotide sequenceof the isolated human Δ5-desaturase gene is shown in FIG. 12 (SEQ IDNO:1), and the amino acid sequence of the corresponding purified proteinis shown in FIG. 13 (SEQ ID NO:12).

As an example, the isolated human Δ5-desaturase gene of the presentinvention converts DGLA to AA or converts 20:4n-3 to EPA. Thus, neitherAA nor EPA, for example, can be synthesized without the Δ5-desaturasegene (e.g., human or M. alpina) and enzyme encoded thereby.

It should be noted that the present invention also encompassesnucleotide sequences (and the corresponding encoded proteins) havingsequences corresponding to or complementary to at least about 50%,preferably at least about 60%, and more preferably at least about 70% ofthe nucleotides in sequence to SEQ ID NO:1 (i.e., the nucleotidesequence of the human Δ5-desaturase gene described herein (see FIG.12)). Such sequences may be derived from non-human sources (e.g., C.elegans or mouse). Furthermore, the present invention also encompassesfragments and derivatives of the nucleotide sequence of the presentinvention (i.e., SEQ ID NO:1), as well as of the sequences derived fromnon-human sources, and having the above-described complementarity orcorrespondence. Functional equivalents of the above-sequences (i.e.,sequences having human Δ5-desaturase activity) are also encompassed bythe present invention. The invention also includes a purifiedpolypeptide which desaturates polyunsaturated fatty acids at the carbon5 position and has at least about 50% amino acid similarity to the aminoacid sequence of the above-noted proteins which are, in turn, encoded bythe above-described nucleotide sequences.

The present invention also encompasses an isolated nucleotide sequencewhich encodes PUFA desaturase activity and that is hybridizable, undermoderately stringent conditions, to a nucleic acid having a nucleotidesequence corresponding to or complementary to the nucleotide sequencerepresented by SEQ ID NO:1 and shown in FIG. 12. A nucleic acid moleculeis “hybridizable” to another nucleic acid molecule when asingle-stranded form of the nucleic acid molecule can anneal to theother nucleic acid molecule under the appropriate conditions oftemperature and ionic strength (see Sambrook et al., “Molecular Cloning:A Laboratory Manual, Second Edition (1989), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.)). The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. “Hybridization” requires that two nucleic acids containcomplementary sequences. However, depending on the stringency of thehybridization, mismatches between bases may occur. The appropriatestringency for hybridizing nucleic acids depends on the length of thenucleic acids and the degree of complementation. Such variables are wellknown in the art. More specifically, the greater the degree ofsimilarity or homology between two nucleotide sequences, the greater thevalue of Tm for hybrids of nucleic acids having those sequences. Forhybrids of greater than 100 nucleotides in length, equations forcalculating Tm have been derived (see Sambrook et al., supra). Forhybridization with shorter nucleic acids, the position of mismatchesbecomes more important, and the length of the oligonucleotide determinesits specificity (see Sambrook et al., supra).

Production of the Human Δ5-Desaturase Enzyme

Once the gene encoding the human Δ5-desaturase enzyme has been isolated,it may then be introduced into either a prokaryotic or eukaryotic hostcell through the use of a vector or construct.

The vector, for example, a bacteriophage, cosmid or plasmid, maycomprise the nucleotide sequence encoding the human Δ5-desaturase enzymeas well as any promoter which is functional in the host cell and is ableto elicit expression of the human Δ5-desaturase encoded by thenucleotide sequence. The promoter is in operable association with oroperably linked to the nucleotide sequence. (A promoter is said to be“operably linked” with a coding sequence if the promoter affectstranscription or expression of the coding sequence.) Suitable promotersinclude, for example, those from genes encoding alcohol dehydrogenase,glyceraldehyde-3-phosphate dehydrogenase, phosphoglucoisomerase,phosphoglycerate kinase, acid phosphatase, T7, TPI, lactase,metallothionein, cytomegalovirus immediate early, whey acidic protein,glucoamylase, and promoters activated in the presence of galactose, forexample, GAL1 and GAL10. Additionally, nucleotide sequences which encodeother proteins, oligosaccharides, lipids, etc. may also be includedwithin the vector as well as other regulatory sequences such as apolyadenylation signal (e.g., the poly-A signal of SV-40T-antigen,ovalalbumin or bovine growth hormone). The choice of sequences presentin the construct is dependent upon the desired expression products aswell as the nature of the host cell.

As noted above, once the vector has been constructed, it may then beintroduced into the host cell of choice by methods known to those ofordinary skill in the art including, for example, transfection,transformation and electroporation (see Molecular Cloning: A LaboratoryManual, 2^(nd) ed., Vol. 1-3, ed. Sambrook et al., Cold Spring HarborLaboratory Press (1989)). The host cell is then cultured under suitableconditions permitting expression of the desired PUFA which is thenrecovered and purified.

Examples of suitable prokaryotic host cells include, for example,bacteria such as Escherichia coli, Bacillus subtilis as well ascyanobacteria such as Spirulina spp. (i.e., blue-green algae). Examplesof suitable eukaryotic host cells include, for example, mammalian cells,plant cells, yeast cells such as Saccharomyces cerevisiae, Saccharomycescarlsbergensis, Lipomyces starkey, Candida spp. such as Yarrowia(Candida) lipolytica, Kluyveromyces spp., Pichia spp., Trichoderma spp.or Hansenula spp., or fungal cells such as filamentous fungal cells, forexample, Aspergillus, Neurospora and Penicillium. Preferably,Saccharomyces cerevisiae (baker's yeast) cells are utilized.

Expression in a host cell can be accomplished in a transient or stablefashion. Transient expression can occur from introduced constructs whichcontain expression signals functional in the host cell, but whichconstructs do not replicate and rarely integrate in the host cell, orwhere the host cell is not proliferating. Transient expression also canbe accomplished by inducing the activity of a regulatable promoteroperably linked to the gene of interest, although such inducible systemsfrequently exhibit a low basal level of expression. Stable expressioncan be achieved by introduction of a construct that can integrate intothe host genome or that autonomously replicates in the host cell. Stableexpression of the gene of interest can be selected for through the useof a selectable marker located on or transfected with the expressionconstruct, followed by selection for cells expressing the marker. Whenstable expression results from integration, the site of the construct'sintegration can occur randomly within the host genome or can be targetedthrough the use of constructs containing regions of homology with thehost genome sufficient to target recombination with the host locus.Where constructs are targeted to an endogenous locus, all or some of thetranscriptional and translational regulatory regions can be provided bythe endogenous locus.

A transgenic mammal may also be used in order to express the enzyme ofinterest (i.e., the human Δ5-desaturase), and ultimately the PUFA(s) ofinterest. More specifically, once the above-described construct iscreated, it may be inserted into the pronucleus of an embryo. The embryomay then be implanted into a recipient female. Alternatively, a nucleartransfer method could also be utilized (Schnieke et al., Science278:2130-2133 (1997)). Gestation and birth are then permitted (see,e.g., U.S. Pat. No. 5,750,176 and U.S. Pat. No. 5,700,671). Milk, tissueor other fluid samples from the offspring should then contain alteredlevels of PUFAs, as compared to the levels normally found in thenon-transgenic animal. Subsequent generations may be monitored forproduction of the altered or enhanced levels of PUFAs and thusincorporation of the gene encoding the human Δ5-desaturase enzyme intotheir genomes. The mammal utilized as the host may be selected from thegroup consisting of, for example, a mouse, a rat, a rabbit, a pig, agoat, a sheep, a horse and a cow. However, any mammal may be usedprovided it has the ability to incorporate DNA encoding the enzyme ofinterest into its genome.

For expression of a human Δ5-desaturase polypeptide, functionaltranscriptional and translational initiation and termination regions areoperably linked to the DNA encoding the desaturase polypeptide.Transcriptional and translational initiation and termination regions arederived from a variety of nonexclusive sources, including the DNA to beexpressed, genes known or suspected to be capable of expression in thedesired system, expression vectors, chemical synthesis, or from anendogenous locus in a host cell. Expression in a plant tissue and/orplant part presents certain efficiencies, particularly where the tissueor part is one which is harvested early, such as seed, leaves, fruits,flowers, roots, etc. Expression can be targeted to that location withthe plant by utilizing specific regulatory sequence such as those ofU.S. Pat. Nos. 5,463,174, 4,943,674, 5,106,739, 5,175,095, 5,420,034,5,188,958, and 5,589,379. Alternatively, the expressed protein can be anenzyme which produces a product which may be incorporated, eitherdirectly or upon further modifications, into a fluid fraction from thehost plant. Expression of a human Δ5-desaturase gene, or antisense humanΔ5-desaturase transcripts, can alter the levels of specific PUFAs, orderivatives thereof, found in plant parts and/or plant tissues. Thehuman Δ5-desaturase polypeptide coding region may be expressed either byitself or with other genes, in order to produce tissues and/or plantparts containing higher proportions of desired PUFAs or in which thePUFA composition more closely resembles that of human breast milk(Prieto et al., PCT publication WO 95/24494). The termination region maybe derived from the 3′ region of the gene from which the initiationregion was obtained or from a different gene. A large number oftermination regions are known to and have been found to be satisfactoryin a variety of hosts from the same and different genera and species.The termination region usually is selected as a matter of conveniencerather than because of any particular property.

As noted above, a plant (e.g., Glycine max (soybean) or Brassica napus(canola)) or plant tissue may also be utilized as a host or host cell,respectively, for expression of the human Δ5-desaturase enzyme whichmay, in turn, be utilized in the production of polyunsaturated fattyacids. More specifically, desired PUFAS can be expressed in seed.Methods of isolating seed oils are known in the art. Thus, in additionto providing a source for PUFAs, seed oil components may be manipulatedthrough the expression of the human Δ5-desaturase gene, as well asperhaps other desaturase genes and elongase genes, in order to provideseed oils that can be added to nutritional compositions, pharmaceuticalcompositions, animal feeds and cosmetics. Once again, a vector whichcomprises a DNA sequence encoding the human Δ5-desaturase operablylinked to a promoter, will be introduced into the plant tissue or plantfor a time and under conditions sufficient for expression of the humanΔ5-desaturase gene. The vector may also comprise one or more genes thatencode other enzymes, for example, Δ4-desaturase, elongase,Δ6-desaturase, Δ12-desaturase, Δ15-desaturase, Δ17-desaturase, and/orΔ19-desaturase. The plant tissue or plant may produce the relevantsubstrate (e.g., DGLA, GLA, EPA, 20:4n-3, etc.) upon which the enzymesact or a vector encoding enzymes which produce such substrates may beintroduced into the plant tissue, plant cell or plant. In addition,substrate may be sprayed on plant tissues expressing the appropriateenzymes. Using these various techniques, one may produce PUFAs (e.g.,n-6 unsaturated fatty acids such as AA, or n-3 fatty acids such as EPAor DHA) by use of a plant cell, plant tissue or plant. It should also benoted that the invention also encompasses a transgenic plant comprisingthe above-described vector, wherein expression of the nucleotidesequence of the vector results in production of a polyunsaturated fattyacid in, for example, the seeds of the transgenic plant.

The substrates which may be produced by the host cell either naturallyor transgenically, as well as the enzymes which may be encoded by DNAsequences present in the vector which is subsequently introduced intothe host cell, are shown in FIG. 11.

In view of the above, the present invention encompasses a method ofproducing the human Δ5-desaturase enzyme comprising the steps of: 1)isolating the nucleotide sequence of the gene encoding humanΔ5-desaturase enzyme; 2) constructing a vector comprising saidnucleotide sequence; and 3) introducing said vector into a host cellunder time and conditions sufficient for the production of thedesaturase enzyme.

The present invention also encompasses a method of producingpolyunsaturated fatty acids comprising exposing an acid to the humanΔ5-desaturase enzyme such that the desaturase converts the acid to apolyunsaturated fatty acid. For example, when 20:3n-6 is exposed tohuman Δ5-desaturase enzyme, it is converted to AA. AA may then beexposed to elongase which elongates the AA to adrenic acid (i.e.,22:4n-6). Alternatively, human Δ5-desaturase may be utilized to convert20:4n-3 to 20:5n-3 which may be exposed to elongase and converted to(n-3)-docosapentaenoic acid. The (n-3)-docosapentaenoic acid may then beconverted to DHA by use of Δ4-desaturase. Thus, human Δ5-desaturase maybe used in the production of polyunsaturated fatty acids which may beused, in turn, for particular beneficial purposes.

Uses of the Human Δ5-Desaturase Gene and Enzyme Encoded Thereby

As noted above, the isolated human Δ5-desaturase gene and the desaturaseenzyme encoded thereby have many uses. For example, the gene andcorresponding enzyme may be used indirectly or directly in theproduction of polyunsaturated fatty acids, for example, AA, adrenic acidor EPA. (“Directly” is meant to encompass the situation where the enzymedirectly converts the acid to another acid, the latter of which isutilized in a composition (e.g., the conversion of DGLA to AA).“Indirectly” is meant to encompass the situation where an acid isconverted to another acid (i.e., a pathway intermediate) by thedesaturase (e.g., DGLA to AA) and then the latter acid is converted toanother acid by use of a non-desaturase enzyme (e.g., AA to adrenic acidby elongase or by use of another desaturase enzyme (e.g., AA to EPA byΔ17-desaturase.)). These polyunsaturated fatty acids (i.e., thoseproduced either directly or indirectly by activity of the desaturaseenzyme) may be added to, for example, nutritional compositions,pharmaceutical compositions, cosmetics, and animal feeds, all of whichare encompassed by the present invention. These uses are described, indetail, below.

Nutritional Compositions

The present invention includes nutritional compositions. Suchcompositions, for purposes of the present invention, include any food orpreparation for human consumption including for enteral or parenteralconsumption, which when taken into the body (a) serve to nourish orbuild up tissues or supply energy and/or (b) maintain, restore orsupport adequate nutritional status or metabolic function.

The nutritional composition of the present invention comprises at leastone oil or acid produced directly or indirectly by use of the humanΔ5-desaturase gene, in accordance with the present invention, and mayeither be in a solid or liquid form. Additionally, the composition mayinclude edible macronutrients, vitamins and minerals in amounts desiredfor a particular use. The amount of such ingredients will vary dependingon whether the composition is intended for use with normal, healthyinfants, children or adults having specialized needs such as those whichaccompany certain metabolic conditions (e.g., metabolic disorders).

Examples of macronutrients which may be added to the composition includebut are not limited to edible fats, carbohydrates and proteins. Examplesof such edible fats include but are not limited to coconut oil, soy oil,and mono- and diglycerides. Examples of such carbohydrates include butare not limited to glucose, edible lactose and hydrolyzed search.Additionally, examples of proteins which may be utilized in thenutritional composition of the invention include but are not limited tosoy proteins, electrodialysed whey, electrodialysed skim milk, milkwhey, or the hydrolysates of these proteins.

With respect to vitamins and minerals, the following may be added to thenutritional compositions of the present invention: calcium, phosphorus,potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc,selenium, iodine, and Vitamins A, E, D, C, and the B complex. Other suchvitamins and minerals may also be added.

The components utilized in the nutritional compositions of the presentinvention will be of semi-purified or purified origin. By semi-purifiedor purified is meant a material which has been prepared by purificationof a natural material or by synthesis.

Examples of nutritional compositions of the present invention includebut are not limited to infant formulas, dietary supplements, dietarysubstitutes, and rehydration compositions. Nutritional compositions ofparticular interest include but are not limited to those utilized forenteral and parenteral supplementation for infants, specialist infantformulas, supplements for the elderly, and supplements for those withgastrointestinal difficulties and/or malabsorption.

The nutritional composition of the present invention may also be addedto food even when supplementation of the diet is not required. Forexample, the composition may be added to food of any type including butnot limited to margarines, modified butters, cheeses, milk, yogurt,chocolate, candy, snacks, salad oils, cooking oils, cooking fats, meats,fish and beverages.

In a preferred embodiment of the present invention, the nutritionalcomposition is an enteral nutritional product, more preferably, an adultor pediatric enteral nutritional product. This composition may beadministered to adults or children experiencing stress or havingspecialized needs due to chronic or acute disease states. Thecomposition may comprise, in addition to polyunsaturated fatty acidsproduced in accordance with the present invention, macronutrients,vitamins and minerals as described above. The macronutrients may bepresent in amounts equivalent to those present in human milk or on anenergy basis, i.e., on a per calorie basis.

Methods for formulating liquid or solid enteral and parenteralnutritional formulas are well known in the art. (See also the Examplesbelow.)

The enteral formula, for example, may be sterilized and subsequentlyutilized on a ready-to-feed (RTF) basis or stored in a concentratedliquid or powder. The powder can be prepared by spray drying the formulaprepared as indicated above, and reconstituting it by rehydrating theconcentrate. Adult and pediatric nutritional formulas are well known inthe art and are commercially available (e.g., Similac®, Ensure®, Jevity®and Alimentum® from Ross Products Division, Abbott Laboratories,Columbus, Ohio). An oil or acid produced in accordance with the presentinvention may be added to any of these formulas.

The energy density of the nutritional compositions of the presentinvention, when in liquid form, may range from about 0.6 Kcal to about 3Kcal per ml. When in solid or powdered form, the nutritional supplementsmay contain from about 1.2 to more than 9 Kcals per gram, preferablyabout 3 to 7 Kcals per gm. In general, the osmolality of a liquidproduct should be less than 700 mOsm and, more preferably, less than 660mOsm.

The nutritional formula may include macronutrients, vitamins, andminerals, as noted above, in addition to the PUFAs produced inaccordance with the present invention. The presence of these additionalcomponents helps the individual ingest the minimum daily requirements ofthese elements. In addition to the provision of PUFAs, it may also bedesirable to add zinc, copper, folic acid and antioxidants to thecomposition. It is believed that these substance boost a stressed immunesystem and will therefore provide further benefits to the individualreceiving the composition. A pharmaceutical composition may also besupplemented with these elements.

In a more preferred embodiment, the nutritional composition comprises,in addition to antioxidants and at least one PUFA, a source ofcarbohydrate wherein at least 5 weight percent of the carbohydrate isindigestible oligosaccharide. In a more preferred embodiment, thenutritional composition additionally comprises protein, taurine, andcarnitine.

As noted above, the PUFAs produced in accordance with the presentinvention, or derivatives thereof, may be added to a dietary substituteor supplement, particularly an infant formula, for patients undergoingintravenous feeding or for preventing or treating malnutrition or otherconditions or disease states. As background, it should be noted thathuman breast milk has a fatty acid profile comprising from about 0.15%to about 0.36% as DHA, from about 0.03% to about 0.13% as EPA, fromabout 0.30% to about 0.88% as AA, from about 0.22% to about 0.67% asDGLA, and from about 0.27% to about 1.04% as GLA. Thus, fatty acids suchas AA, EPA and/or docosahexaenoic acid (DHA), produced in accordancewith the present invention, can be used to alter, for example, thecomposition of infant formulas in order to better replicate the PUFAcontent of human breast milk or to alter the presence of PUFAs normallyfound in a non-human mammal's milk. In particular, a composition for usein a pharmacologic or food supplement, particularly a breast milksubstitute or supplement, will preferably comprise one or more of AA,DGLA and GLA. More preferably, the oil will comprise from about 0.3 to30% AA, from about 0.2 to 30% DGLA, and/or from about 0.2 to about 30%GLA.

Parenteral nutritional compositions comprising from about 2 to about 30weight percent fatty acids calculated as triglycerides are encompassedby the present invention. The preferred composition has about 1 to about25 weight percent of the total PUFA composition as GLA (U.S. Pat. No.5,196,198). Other vitamins, particularly fat-soluble vitamins such asvitamin A, D, E and L-carnitine can optionally be included. Whendesired, a preservative such as alpha-tocopherol may be added in anamount of about 0.1% by weight.

In addition, the ratios of AA, DGLA and GLA can be adapted for aparticular given end use. When formulated as a breast milk supplement orsubstitute, a composition which comprises one or more of AA, DGLA andGLA will be provided in a ratio of about 1:19:30 to about 6:1:0.2,respectively. For example, the breast milk of animals can vary in ratiosof AA:DGLA:GLA ranging from 1:19:30 to 6:1:0.2, which includesintermediate ratios which are preferably about 1:1:1, 1:2:1, 1:1:4. Whenproduced together in a host cell, adjusting the rate and percent ofconversion of a precursor substrate such as GLA and DGLA to AA can beused to precisely control the PUFA ratios. For example, a 5% to 10%conversion rate of DGLA to AA can be used to produce an AA to DGLA ratioof about 1:19, whereas a conversion rate of about 75% TO 80% can be usedto produce an AA to DGLA ratio of about 6:1. Therefore, whether in acell culture system or in a host animal, regulating the timing, extentand specificity of human Δ5-desaturase expression, as well as theexpression of other desaturases and elongases, can be used to modulatePUFA levels and ratios. The PUFAs/acids produced in accordance with thepresent invention (e.g., AA and EPA) may then be combined with otherPUFAs/acids (e.g., GLA) in the desired concentrations and ratios.

Additionally, PUFA produced in accordance with the present invention orhost cells containing them may also be used as animal food supplementsto alter an animal's tissue or milk fatty acid composition to one moredesirable for human or animal consumption.

Pharmaceutical Compositions

The present invention also encompasses a pharmaceutical compositioncomprising one or more of the acids and/or resulting oils produced usingthe human Δ5-desaturase gene, in accordance with the methods describedherein. More specifically, such a pharmaceutical composition maycomprise one or more of the acids and/or oils as well as a standard,well-known, non-toxic pharmaceutically acceptable carrier, adjuvant orvehicle such as, for example, phosphate buffered saline, water, ethanol,polyols, vegetable oils, a wetting agent or an emulsion such as awater/oil emulsion. The composition may be in either a liquid or solidform. For example, the composition may be in the form of a tablet,capsule, ingestible liquid or powder, injectible, or topical ointment orcream. Proper fluidity can be maintained, for example, by themaintenance of the required particle size in the case of dispersions andby the use of surfactants. It may also be desirable to include isotonicagents, for example, sugars, sodium chloride and the like. Besides suchinert diluents, the composition can also include adjuvants, such aswetting agents, emulsifying and suspending agents, sweetening agents,flavoring agents and perfuming agents.

Suspensions, in addition to the active compounds, may comprisesuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanthor mixtures of these substances.

Solid dosage forms such as tablets and capsules can be prepared usingtechniques well known in the art. For example, PUFAs produced inaccordance with the present invention can be tableted with conventionaltablet bases such as lactose, sucrose, and cornstarch in combinationwith binders such as acacia, cornstarch or gelatin, disintegratingagents such as potato starch or alginic acid, and a lubricant such asstearic acid or magnesium stearate. Capsules can be prepared byincorporating these excipients into a gelatin capsule along withantioxidants and the relevant PUFA(s). The antioxidant and PUFAcomponents should fit within the guidelines presented above.

For intravenous administration, the PUFAs produced in accordance withthe present invention or derivatives thereof may be incorporated intocommercial formulations such as Intralipids™. The typical normal adultplasma fatty acid profile comprises 6.64 to 9.46% of AA, 1.45 to 3.11%of DGLA, and 0.02 to 0.08% of GLA. These PUFAs or their metabolicprecursors can be administered alone or in combination with other PUFAsin order to achieve a normal fatty acid profile in a patient. Wheredesired, the individual components of the formulations may be providedindividually, in kit form, for single or multiple use. A typical dosageof a particular fatty acid is from 0.1 mg to 20 g (up to 100 g) dailyand is preferably from 10 mg to 1, 2, 5 or 10 g daily.

Possible routes of administration of the pharmaceutical compositions ofthe present invention include, for example, enteral (e.g., oral andrectal) and parenteral. For example, a liquid preparation may beadministered, for example, orally or rectally. Additionally, ahomogenous mixture can be completely dispersed in water, admixed understerile conditions with physiologically acceptable diluents,preservatives, buffers or propellants in order to form a spray orinhalant.

The route of administration will, of course, depend upon the desiredeffect. For example, if the composition is being utilized to treatrough, dry, or aging skin, to treat injured or burned skin, or to treatskin or hair affected by a disease or condition, it may perhaps beapplied topically.

The dosage of the composition to be administered to the patient may bedetermined by one of ordinary skill in the art and depends upon variousfactors such as weight of the patient, age of the patient, immune statusof the patient, etc.

With respect to form, the composition may be, for example, a solution, adispersion, a suspension, an emulsion or a sterile powder which is thenreconstituted.

The present invention also includes the treatment of various disordersby use of the pharmaceutical and/or nutritional compositions describedherein. In particular, the compositions of the present invention may beused to treat restenosis after angioplasty. Furthermore, symptoms ofinflammation, rheumatoid arthritis, asthma and psoriasis may also betreated with the compositions of the invention. Evidence also indicatesthat PUFAs may be involved in calcium metabolism; thus, the compositionsof the present invention may, perhaps, be utilized in the treatment orprevention of osteoporosis and of kidney or urinary tract stones.

Additionally, the compositions of the present invention may also be usedin the treatment of cancer. Malignant cells have been shown to havealtered fatty acid compositions. Addition of fatty acids has been shownto slow their growth, cause cell death and increase their susceptibilityto chemotherapeutic agents. Moreover, the compositions of the presentinvention may also be useful for treating cachexia associated withcancer.

The compositions of the present invention may also be used to treatdiabetes (see U.S. Pat. No. 4,826,877 and Horrobin et al., Am. J. Clin.Nutr. Vol. 57 (Suppl.) 732S-737S). Altered fatty acid metabolism andcomposition have been demonstrated in diabetic animals.

Furthermore, the compositions of the present invention, comprising PUFAsproduced either directly or indirectly through the use of the humanΔ5-desaturase enzyme, may also be used in the treatment of eczema, inthe reduction of blood pressure, and in the improvement of mathematicsexamination scores. Additionally, the compositions of the presentinvention may be used in inhibition of platelet aggregation, inductionof vasodilation, reduction in cholesterol levels, inhibition ofproliferation of vessel wall smooth muscle and fibrous tissue (Brenneret al., Adv. Exp. Med. Biol. Vol. 83, p.85-101, 1976), reduction orprevention of gastrointestinal bleeding and other side effects ofnon-steroidal anti-inflammatory drugs (see U.S. Pat. No. 4,666,701),prevention or treatment of endometriosis and premenstrual syndrome (seeU.S. Pat. No. 4,758,592), and treatment of myalgic encephalomyelitis andchronic fatigue after viral infections (see U.S. Pat. No. 5,116,871).

Further uses of the compositions of the present invention include use inthe treatment of AIDS, multiple sclerosis, and inflammatory skindisorders, as well as for maintenance of general health.

Additionally, the composition of the present invention may be utilizedfor cosmetic purposes. It may be added to pre-existing cosmeticcompositions such that a mixture is formed or may be used as a solecomposition.

Veterinary Applications

It should be noted that the above-described pharmaceutical andnutritional compositions may be utilized in connection with animals(i.e., domestic or non-domestic), as well as humans, as animalsexperience many of the same needs and conditions as humans. For example,the oil or acids of the present invention may be utilized in animal feedsupplements, animal feed substitutes, animal vitamins or in animaltopical ointments.

The present invention may be illustrated by the use of the followingnon-limiting examples:

EXAMPLE I Human Desaturase Gene Sequences

As described in International Application PCT/US98/07422 (hereinincorporated in its entirety by reference), the putative humandesaturase gene sequences involved in long chain polyunsaturated fattyacid biosynthesis were isolated based on homology between the human cDNAsequences and Mortierella alpina desaturase gene sequences. The threeconserved “histidine boxes” known to be conserved among membrane-bounddesaturases were found. As with other membrane-bound desaturases, thefinal HXXHH histidine box motif was found to be QXXHH. The amino acidsequence of the putative human desaturases exhibited homology to M.alpina Δ5-, Δ6-, Δ9-, and Δ12-desaturases.

The M. alpina Δ5-desaturase and Δ6-desaturase cDNA sequences were usedto search the LifeSeq database of Incyte Pharmaceuticals, Inc., PaloAlto, Calif. The Δ5-desaturase sequence was divided into fragments: 1)amino acid no. 1-150, 2) amino acid no. 151-300, and 3) amino acid no.301-446. The Δ6 desaturase sequence was divided into three fragments: 1)amino acid no. 1-150, 2) amino acid no. 151-300, and 3) amino acid no.301-457. These polypeptide fragments were searched against the databaseusing the “tblastn” algorithm. This algorithm compares a protein querysequence against a nucleotide sequence database dynamically translatedin all six reading frames (both strands).

The polypeptide fragments 2 and 3 of M. alpina Δ5- and Δ6-desaturaseshave homologies with the CloneID sequences as outlined in FIG. 1. TheCloneID represents an individual sequence from the Incyte LifeSeqdatabase. After the “tblastn” results had been reviewed, CloneInformation was searched with the default settings of Stringencyof >=50, and Productscore <=100 for different CloneID numbers. The CloneInformation Results displayed the information including the ClusterID,CloneID, Library, HitID, and Hit Description. When selected, theClusterID number displayed the clone information of all the clones thatbelong in that ClusterID. The Assemble command assembled all of theCloneID which comprise the ClusterID. The following default setting wereused for GCG (Genetics Computer Group, University of WisconsinBiotechnology Center, Madison, Wis.) Assembly:

Word Size: 7; Minimum Overlap: 14; Stringency: 0.8;

Minimum Identity: 14; Maximum Gap: 10; Gap Weight: 8; and

Length Weight: 2.

GCG Assembly Results displayed the contigs generated on the basis ofsequence information within the CloneID. A contig is an alignment of DNAsequences based on areas of homology among these sequences. A newsequence (consensus sequence) was generated based on the aligned DNAsequence within a contig. The contig. containing the CloneID wasidentified, and the ambiguous sites of the consensus sequence wereedited based on the alignment of the CloneIDs (see FIGS. 2-6) togenerate the best possible sequence. The procedure was repeated for allsix CloneID listed in FIG. 1. This produced five unique contigs. Theedited consensus sequences of the 5 contigs were imported into theSequencher software program (Gene Codes Corporation, Ann Arbor, Mich.).These consensus sequences were assembled. The contig 2511785 overlapswith contig 3506132, and this new contig was called 2535 (FIG. 7). Thecontigs from the Sequencher program were copied into the SequenceAnalysis software package of GCG.

Each contig was translated in all six reading frames into proteinsequences. The M. alpina Δ5-desaturase (Ma29) and Δ6-desaturase (Ma524)sequences were compared with each of the translated contigs using theFastA search (a Pearson and Lipman search for similarity between a querysequence and a group of sequences of the same type (nucleic acid orprotein)). Homology among these sequences suggest the open readingframes of each contig as underlined in FIGS. 3, 5, and 7. The homologyamong the M. alpina Δ5- and Δ6-desaturase sequences to contigs 2535 and3854933 were utilized to create the final contig called 253538a (seeFIG. 8). FIG. 9 is the FastA match of the translated sequences of thefinal contig 253538a and Ma29, and FIG. 10 is the FastA match of thetranslated sequences of the final contig 253538a and Ma524.

Although the open reading frame was generated by merging the twocontigs, the contig 2535 shows that there is a unique sequence in thebeginning of this contig which does not match with the contig 3854933.Therefore, it is possible that these contigs were generated fromindependent desaturase-like human genes.

The contig 253538a contains an open reading frame encoding 432 aminoacid (FIG. 8, underlined). It starts with Gln (CAG) and ends with thestop codon (TGA) (both in bold). The contig 253538a aligns with both M.alpina Δ5- and Δ6-desaturase sequences, suggesting that it could beeither of the desaturases, as well as other known desaturases whichshare homology with each other. The individual contigs listed in FIG. 1,as well as the intermediate contig 2535 and the final contig 253538a canbe utilized to isolate the complete genes for human desaturases.

Determination of Human Δ5-Desaturase Gene Sequence

Primers RO384 and RO388 were designed based on the 5′ and 3′ sequences,respectively, of contig 2535. The human monocyte cDNA library (Clontech,Palo Alto, Calif.) was amplified with the vector primer RO329 (5′-CAGACC AAC TGG TAA TGG TAG-3′) and RO384 (5′-TCA GGC CCA AGC TGG ATG GCTGCA ACA TG-3′), and also with the vector primer RO328 (5′-CTC CTG GAGCCC GTC AGT ATC-3′) and RO388 (5′-ATG GTG GGG AAG AGG TGG TGC TCA ATCTG-3′). Polymerase Chain Reaction (PCR) was carried out in a 100 μlvolume containing: 1 μl of human monocyte cDNA library, 10 pM eachprimer, 10 μl of 10× buffer and 1.0 U of Taq Polymerase. Thermocyclerconditions in Perkin Elmer 9600 were as follows:

-   94° C. for 2 mins, then 30 cycles of 94° C. for 1 min., 58° C. for 2    mins. and 72° C. for 3 mins. PCR was followed by an additional    extension at 72° C. for 7 minutes.

The PCR amplified mixture was run on a gel, and the amplified fragmentswere gel purified. The isolated fragment from PCR amplification withRO329 and RO384 was approximately 900 bp, and that from PCRamplification with RO328 and RO388 was approximately 650 bp. Theseisolated fragments were filled-in using T4 DNA polymerase, and thefilled-in fragments were cloned into the PCR-Blunt vector (InvitrogenCorp., Carlsbad, Calif.). The clone of RO329/RO384 amplified fragmentwas designated as pRAE-7, and the clone of RO328/RO388 amplifiedfragment was designated as pRAE-8. Both ends of the clones weresequenced using ABI 373 DNA Sequencer (Applied Biosystems, Foster City,Calif.) and assembled using the Sequencher program (a sequence analysisprogram, Gene Codes Corporation, Ann Arbor, Mich.). This assembly of thesequences revealed that the two clones contained different sizes of thesame gene (FIG. 14). The complete sequence of the pRAE-7 gene wascompiled (FIG. 15) and searched against the known sequences in thepublic database.

The FastA algorithm is a Pearson and Lipman search for similaritybetween a query sequence and a group of sequences of the same type(nucleic acid or protein). The pRAE-7 gene sequence was translated insix reading frames, and using this method, the Swissprot database(Genetics Computer Group (GCG) (Madison, Wis.) was searched. The gene inpRAE-7 was identified as a putative human desaturase based on itshomology to known desaturases. The Swissprot database search producedmatches against the omega-3 fatty acid desaturase from mung bean (23.4%identity in 303 AA overlap), linoleoyl-CoA desaturase from Synechocystissp. (24.3% identity in 280 AA overlap), omega-6 fatty acid desaturasefrom soybean (19.7% identity in 284 AA overlap), and acyl-CoA desaturase1 from Saccharomyces cerevisiae (21.6% identity in 134 AA overlap). TheFastA search against the M. alpina desaturases produced matches againstthe Δ6-(31.9% identity in 285 AA overlap), the Δ5-(28.4% identity in 292AA overlap), and the Δ12-(23.0% identity in 274 AA overlap) desaturases.The matched sequence alignment of the putative human desaturase gene inpRAE-7 against M. alpina Δ5-desaturase (Ma29), M. alpina Δ6-desaturase(Ma524) as well as to the contigs 2535 and 38 are displayed in FIGS. 16,17, 18, and 19 respectively.

The contigs 2535, 38, and 253538a were generated based on assemblies ofvarious sequences as well as their homologies against the knowndesaturases. However, upon examining FIGS. 18 and 19, it can beconcluded that the contigs are merely indications as to what thesequences of the human desaturases might possibly be.

The 5′ end of the gene, the ATG (Methionine), is necessary forexpressing the human desaturase in yeast. FIGS. 16 and 17 show thatpRAE-7 is probably just the last ⅔ of a desaturase gene. Several of theomega-3 and omega-6 fatty acid desaturases, as well as the linoleoyl-CoAdesaturase mentioned above, are smaller than the M. alpina Δ5- andΔ6-desaturases, ranging in sizes of 359-380 amino acids. It wasconcluded from all of the sequences evaluated thus far that the isolatedgene probably needed anywhere from 180-480 bp (60-160 amino acids) ofadditional 5′ sequence for expressing a complete enzyme.

In order to extend the 5′ sequence of the human desaturase gene, theMarathon cDNA Amplification Kit (Clontech, Palo Alto, Calif.) was usedto screen the human liver marathon ready cDNA (Clontech). The rapidamplification of cDNA ends (RACE) reactions are efficient for both 5′and 3′ long-distance PCR. Following the 5′ RACE protocol outlined in thekit, the primers RO430 (5′-GTG GCT GTT GTT ATT GGT GAA GAT AGG CAT C-3′)(designed based on the pRAE-7 gene 3′ sequence, downstream of the TAA(stop)) and the marathon adaptor primer (AP1) from the kit, were used togenerate three PCR amplified products, which were designated A, B, andC. The fragment sizes were approximately 1.5 Kb, 1.4 Kb, 1.2 Kb,respectively. The fragments were filled-in with T4 DNA polymerase, andcloned into the pCR-blunt vector. A total of twenty-two clones weregenerated and sequenced. Using the FastA algorithm, the sequences weresearched against the GenEMBL database of GCG.

Many of the sequences had a great homology to the human DNA sequencewith the GenBank accession number of AC004228. This DNA sequence isdescribed as: Sequencing in Progress, Homo sapiens Chromosome 11q12pacpDJ519o3; HTGS phase 1, 18 unordered pieces. The 18 contigs wererecorded in an arbitrary fashion. Using this sequence information andthe information from the assembled sequences of the clones, the cloneswere categorized into five groups.

All of the clones have the same sequence downstream of the BamHI site(see FIG. 12, underlined). But each group represents a different 5′sequence, with a total of 10 clones being too short to be the fulllength gene. Group 1, represented by clone A-1, is comprised of 5 cloneswhich have homology to cytochrome b5 gene (FIG. 20). A translationalstart codon, ATG, is not present in clone A-1; however, as can be seenin FIG. 21, there is an ATG (underlined) present in the ac004228sequence 17 bp upstream of the strong area of homology between A-1 andac004228. Starting from the strong area of homology, A-1 has an openreading frame of 1318 bp. However, starting from the ATG, the openreading frame is 1335 bp. Group 2, represented by clone 3-5, iscomprised of 3 clones which have an ATG within an NcoI site, but fourtranslational stop codons between the ATG and the BamHI site (FIG. 22,the NcoI, BamHI sites are in bold, and the four termination codons areunderlined). Group 3 is comprised of one clone, A-10, which has an ATG135 bp upstream of the BamHI site, giving an open reading frame of 1267bp (FIG. 23). Group 4 is comprised of 2 clones, represented by cloneA-16, which does not have an ATG; however, upstream of where thesequence aligns with ac004228, there is an ATG (FIG. 24, underlined).The open reading frame of this group is 1347 bp. Group 5 is comprised ofone clone which does not have an ATG. However, this clone matches theac004228 sequence even upstream of the BamHI site (FIG. 25).

As illustrated in FIG. 26, many of the clones from the five groups arerepresented in order with the ac004228 sequence. There appeared to be ahigh level of splicing, with the sequence downstream of the BamHI site(in bold) acting as the common anchor for the various 5′ exons. All ofthe potential start sites are also in bold, and the sequences foundwithin the clones have been underlined.

The A-1 sequence was used to search the LifeSeq database of IncytePharmaceuticals, Inc., Palo Alto, Calif., to see if its latest versionwould also have sequences with homology to our desaturase gene sequence.Two contigs were generated in this search, contig 3381584 and contig2153526. The human desaturase gene sequence was initially compiled basedon sequences from Group I clones and ac004228. However, FIG. 12represents the actual DNA sequence of the isolated gene. The Incytecontigs were used to confirm this sequence (see FIGS. 27 and 28). Thehuman desaturase translated sequence, consisting of 445 amino acids(FIG. 13), was also matched with the original contigs 253538a and 38.These alignments are shown in FIGS. 29 and 30, respectively.

The FastA search of the human desaturase gene against the Swissprotdatabase produced matches against the omega-3 fatty acid desaturase frommung bean (22.4% identity in 381 AA overlap), linoleoyl-CoA desaturasefrom Synechocystis Sp. (24.5% identity in 335 AA overlap), omega-6 fattyacid desaturase from soybean (20.3% identity in 290 AA overlap), andacyl-CoA desaturase 1 from Saccharomyces cerevisiae (21.4% identity in168 AA overlap). The FastA search against M. alpina desaturases producedmatches against the Δ6-(30.5% identity in 455 AA overlap), Δ5-(27.5%identity in 455 AA overlap), and Δ12-desaturases (22.5% identity in 382AA overlap). The FastA match of the human desaturase translated sequenceagainst the ma524 (M. alpina Δ6-desaturase) and ma29 (M. alpinaΔ5-desaturase) sequences are shown in FIGS. 31 and 32, respectively.

EXAMPLE II Construction of Clones

New clones were generated based on clones from three of the Groupsmentioned above, clones A-1, A-10, and A-16. Two primers which weremodified with 5′ phosphate, RO526 (5′-CAT GGC CCC CGA CCC GGT GG-3′) andRO527 (5′-GCG GCC ACC GGG TCG GGG GC-3′), were annealed together to forman adaptor. This adaptor which has NcoI and BsaI overhangs, were ligatedwith the A-1 clone, which had been cut with BsaI/HindIII and gelpurified, for 15 min at room temperature. The pYX242(NcoI/HindIII)vector (Novagen, Madison, Wis.) was added to this ligation mixture andallowed to incubate at room temperature for an additional 45 min. Thisproduced a clone designated as pRAE-28-5. (Plasmid pRAE-28-5 wasdeposited with the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209 on Dec. 21, 1998, under the terms ofthe Budapest Treaty, and was accorded ATCC number ______.)

The A-10 clone was PCR amplified with RO512 (5′-GAT TGG GTG CCA TGG GGATGC GGG ATG AAA AGG C-3′) and RO5 (5′-GAA ACA GCT ATG ACC ATG-3′), theamplified product was cut with NcoI and HindIII and gel purified, andthe purified fragment was cloned into pYX242 (NcoI/HindIII). This newclone was designated as pRAE-26-1.

The A-10 clone was also PCR amplified with RO580 (5′-TCC TGC GAA TTC ACCATG AAA AGG CGG GAG AGA G-3′) and RO5, the amplified product was cutwith NcoI and HindIII and gel purified, and the purified fragment wascloned into pYX242 (NcoI/HindIII). This new clone was designated aspRAE-33.

Two primers which were modified with 5′ phosphate, RO578 (5′-CAT GGC TAGGAG AGG CAG CGC AGC CGC GTC TGG AC-3′) and RO579 (5′-CTA GGT CCA GAC GCGGCT GCG CTG CCT CTC CTA GC-3′), were annealed together to form anadaptor. This adaptor which has NcoI and BlnI overhangs, were ligatedwith the A-16 clone, which had been cut with BlnI/HindIII and gelpurified, for 15 min at room temperature. The pYX242(NcoI/HindIII)vector was added to this ligation mixture and allowed to incubate atroom temperature for an additional 45 min. This produced a clonedesignated as pRAE-35.

EXAMPLE III Expression of Human Δ5-Desaturase

The constructs pRAE-26-1, pPAE-28-5, pRAE-33, and pRAE-35 weretransformed into S. cerevisiae 334 and screened for desaturase activity.The substrates DGLA (20:3n-6), OA(18:1n-9), AA(20:4n-6), and LA(18:2n-6)were used to determine the activity of the expressed gene fromconstructs pRAE-26-1 and pRAE-28-5. Only the substrate DGLA was used todetermine the activity of the expressed gene from all of the constructs.The negative control strain was S. cerevisiae 334 containing theunaltered pYX242 vector. The cultures were grown for 48 hours at 30° C.,in selective media (Ausubel et al., Short Protocols in MolecularBiology, Ch. 13, P. 3-5 (1992)), in the presence of a particularsubstrate. Lipid fractions of each culture were extracted for analysis.The desaturase activity results are provided in FIGS. 33 and 34.

All of the values in FIG. 33 are the average of two separate samples perstrain, tested in the same run. The substrate, as well as the fatty acidit was converted to, is shown in bold. The expressed gene in the strain334 (pRAE-28-5) is a Δ5-desaturase. It converted the substrate DGLA to ahigher percent of AA than the control strain 334(pYX242), 0.127% vs.0.062%, respectively. The percent of AA present in the cultures ofstrains 334(pRAE-26-1), 334(pRAE-33), and 334(pRAE-35) are comparable tothat of the control strain (0.075%, 0.062%, and 0.063%, respectively).Therefore, it can be concluded that the cyt b5 sequence containing genein the construct pRAE-28-5 expresses an active human Δ5-desaturase;whereas, the other variations of the gene do not.

The activity of the human Δ5-desaturase was further confirmed in theexperiment outlined in FIG. 34. Included in this figure are the fattyacid profiles of the strains 334 (pRAE-28-5), 334 (pRAE-26-1), and thecontrol strain 334 (pYX242) when DGLA(20:3n-6), OA(18:1n-9),AA(20:4n-6), or LA(18:2n-6) was used as the substrate, as well as whenno substrate was added. Again, the strain 334(pRAE-28-5) expressed anactive human Δ5-desaturase, converting DGLA to AA at a higher percentthan the control strain, 0.106% vs. 0.065%, respectively. The strain334(pRAE-26-1) had about the same amount of AA (0.06%) as the control.The conversion of the substrate OA to LA was not detected, confirmingthat the strains do not have a Δ12-desaturase activity. The conversionof the substrate AA to eicosapentaenoic acid (EPA, 20:5n-3) wasdetected, but at a very low level equal to that of the control strain,confirming that the strains do not have a Δ17-desaturase activity. Theconversion of the substrate LA to GLA was detected, but again at a verylow level equal to the control strain, confirming that the strains donot have a Δ6-desaturase activity.

The present sequence (FIG. 12) differs from the Genbank sequenceg3169158 of the LifeSeq database with respect to two positions. Inparticular, with respect to the nucleotide sequence of sequenceg3169158, position 1082 is an adenosine; however, in the presentsequence position 1082 is a thymine (see FIG. 12). Furthermore, position1229 of sequence g3169158 is an adenine whereas in the present sequenceposition 1229 is a guanine. In terms of an amino acid sequencecomparison, position 361 of the present sequence is a leucine (see FIG.13), and position 361 of sequence g3169158 is a glutamine. Furthermore,position 410 of the present sequence is an arginine, whereas position410 of sequence g3169158 is a histidine. Additionally, sequence g3169158is described, in the database, as a “hypothetical protein” which“exhibits similarity to motifs found in delta 6 desaturase, ahypothetical cytochrome b5 containing fusion protein.” However, asdemonstrated in the above example, the protein encoded by the sequencein FIG. 12 is a human Δ5-desaturase, not a Δ6-desaturase.

Nutritional Compositions

The PUFAs described in the Detailed Description may be utilized invarious nutritional supplements, infant formulations, nutritionalsubstitutes and other nutritional solutions.

I. Infant Formulations

A. Isomil® Soy Formula with Iron:

Usage: As a beverage for infants, children and adults with an allergy orsensitivity to cows milk. A feeding for patients with disorders forwhich lactose should be avoided: lactase deficiency, lactose intoleranceand galactosemia.

Features:

-   -   Soy protein isolate to avoid symptoms of cow's-milk-protein        allergy or sensitivity.    -   Lactose-free formulation to avoid lactose-associated diarrhea.    -   Low osmolality (240 mOs/kg water) to reduce risk of osmotic        diarrhea.    -   Dual carbohydrates (corn syrup and sucrose) designed to enhance        carbohydrate absorption and reduce the risk of exceeding the        absorptive capacity of the damaged gut.    -   1.8 mg of Iron (as ferrous sulfate) per 100 Calories to help        prevent iron deficiency.    -   Recommended levels of vitamins and minerals.    -   Vegetable oils to provide recommended levels of essential fatty        acids.    -   Milk-white color, milk-like consistency and pleasant aroma.        Ingredients: (Pareve) 85% water, 4.9% corn syrup, 2.6% sugar        (sucrose), 2.1% soy oil, 1.9% soy protein isolate, 1.4% coconut        oil, 0.15% calcium citrate, 0.11% calcium phosphate tribasic,        potassium citrate, potassium phosphate monobasic, potassium        chloride, mono- and disglycerides, soy lecithin, carrageenan,        ascorbic acid, L-methionine, magnesium chloride, potassium        phosphate dibasic, sodium chloride, choline chloride, taurine,        ferrous sulfate, m-inositol, alpha-tocopheryl acetate, zinc        sulfate, L-carnitine, niacinamide, calcium pantothenate, cupric        sulfate, vitamin A palmitate, thiamine chloride hydrochloride,        riboflavin, pyridoxine hydrochloride, folic acid, manganese        sulfate, potassium iodide, phylloquinone, biotin, sodium        selenite, vitamin D3 and cyanocobalamin.        B. Isomil® DF Soy Formula for Diarrhea:        Usage: As a short-term feeding for the dietary management of        diarrhea in infants and toddlers.        Features:    -   First infant formula to contain added dietary fiber from soy        fiber specifically for diarrhea management.    -   Clinically shown to reduce the duration of loose, watery stools        during mild to severe diarrhea in infants.    -   Nutritionally complete to meet the nutritional needs of the        infant.    -   Soy protein isolate with added L-methionine meets or exceeds an        infant's requirement for all essential amino acids.    -   Lactose-free formulation to avoid lactose-associated diarrhea.    -   Low osmolality (240 mOsm/kg water) to reduce the risk of osmotic        diarrhea.    -   Dual carbohydrates (corn syrup and sucrose) designed to enhance        carbohydrate absorption and reduce the risk of exceeding the        absorptive capacity of the damaged gut.    -   Meets or exceeds the vitamin and mineral levels recommended by        the Committee on Nutrition of the American Academy of Pediatrics        and required by the Infant Formula Act.    -   1.8 mg of iron (as ferrous sulfate) per 100 Calories to help        prevent iron deficiency.    -   Vegetable oils to provide recommended levels of essential fatty        acids.        Ingredients: (Pareve) 86% water, 4.8% corn syrup, 2.5% sugar        (sucrose), 2.1% soy oil, 2.0% soy protein isolate, 1.4% coconut        oil, 0.77% soy fiber, 0.12% calcium citrate, 0.11% calcium        phosphate tribasic, 0.10% potassium citrate, potassium chloride,        potassium phosphate monobasic, mono and diglycerides, soy        lecithin, carrageenan, magnesium chloride, ascorbic acid,        L-methionine, potassium phosphate dibasic, sodium chloride,        choline chloride, taurine, ferrous sulfate, m-inositol,        alpha-tocopheryl acetate, zinc sulfate, L-carnitine,        niacinamide, calcium pantothenate, cupric sulfate, vitamin A        palmitate, thiamine chloride hydrochloride, riboflavin,        pyridoxine hydrochloride, folic acid, manganese sulfate,        potassium iodide, phylloquinone, biotin, sodium selenite,        vitamin D3 and cyanocobalamin.        C. Isomil® SF Sucrose-Free Soy Formula with Iron:        Usage: As a beverage for infants, children and adults with an        allergy or sensitivity to cow's-milk protein or an intolerance        to sucrose. A feeding for patients with disorders for which        lactose and sucrose should be avoided.        Features:    -   Soy protein isolate to avoid symptoms of cow's-milk-protein        allergy or sensitivity.    -   Lactose-free formulation to avoid lactose-associated diarrhea        (carbohydrate source is Polycose® Glucose Polymers).    -   Sucrose free for the patient who cannot tolerate sucrose.    -   Low osmolality (180 mOsm/kg water) to reduce risk of osmotic        diarrhea.    -   1.8 mg of iron (as ferrous sulfate) per 100 Calories to help        prevent iron deficiency.    -   Recommended levels of vitamins and minerals.    -   Vegetable oils to provide recommended levels of essential fatty        acids.    -   Milk-white color, milk-like consistency and pleasant aroma.        Ingredients: (Pareve) 75% water, 11.8% hydrolized cornstarch,        4.1% soy oil, 4.1% soy protein isolate, 2.8% coconut oil, 1.0%        modified cornstarch, 0.38% calcium phosphate tribasic, 0. 17%        potassium citrate, 0.13% potassium chloride, mono- and        diglycerides, soy lecithin, magnesium chloride, abscorbic acid,        L-methionine, calcium carbonate, sodium chloride, choline        chloride, carrageenan, taurine, ferrous sulfate, m-inositol,        alpha-tocopheryl acetate, zinc sulfate, L-carnitine,        niacinamide, calcium pantothenate, cupric sulfate, vitamin A        palmitate, thiamine chloride hydrochloride, riboflavin,        pyridoxine hydrochloride, folic acid, manganese sulfate,        potassium iodide, phylloquinone, biotin, sodium selenite,        vitamin D3 and cyanocobalamin.        D. Isomil® 20 Soy Formula with Iron Ready to Feed, 20 Cal/fl        oz.:        Usage: When a soy feeding is desired.        Ingredients: (Pareve) 85% water, 4.9% corn syrup, 2.6%        sugar(sucrose), 2.1% soy oil, 1.9% soy protein isolate, 1.4%        coconut oil, 0.15% calcium citrate, 0.11% calcium phosphate        tribasic, potassium citrate, potassium phosphate monobasic,        potassium chloride, mono- and diglycerides, soy lecithin,        carrageenan, abscorbic acid, L-methionine, magnesium chloride,        potassium phosphate dibasic, sodium chloride, choline chloride,        taurine, ferrous sulfate, m-inositol, alpha-tocopheryl acetate,        zinc sulfate, L-carnitine, niacinamide, calcium pantothenate,        cupric sulfate, vitamin A palmitate, thiamine chloride        hydrochloride, riboflavin, pyridoxine hydrochloride, folic acid,        manganese sulfate, potassium iodide, phylloquinone, biotin,        sodium selenite, vitamin D3 and cyanocobalamin.        E. Similac® Infant Formula:        Usage: When an infant formula is needed: if the decision is made        to discontinue breastfeeding before age 1 year, if a supplement        to breastfeeding is needed or as a routine feeding if        breastfeeding is not adopted.        Features:    -   Protein of appropriate quality and quantity for good growth;        heat-denatured, which reduces the risk of milk-associated        enteric blood loss.    -   Fat from a blend of vegetable oils (doubly homogenized),        providing essential linoleic acid that is easily absorbed.    -   Carbohydrate as lactose in proportion similar to that of human        milk.    -   Low renal solute load to minimize stress on developing organs.    -   Powder, Concentrated Liquid and Ready To Feed forms.        Ingredients: (-D) Water, nonfat milk, lactose, soy oil, coconut        oil, mono- and diglycerides, soy lecithin, abscorbic acid,        carrageenan, choline chloride, taurine, m-inositol,        alpha-tocopheryl acetate, zinc sulfate, niacinamide, ferrous        sulfate, calcium pantothenate, cupric sulfate, vitamin A        palmitate, thiamine chloride hydrochloride, riboflavin,        pyridoxine hydrochloride, folic acid, manganese sulfate,        phylloquinone, biotin, sodium selenite, vitamin D3 and        cyanocobalamin.        F. Similac® NeoCare Premature Infant Formula with Iron:        Usage: For premature infants' special nutritional needs after        hospital discharge. Similac NeoCare is a nutritionally complete        formula developed to provide premature infants with extra        calories, protein, vitamins and minerals needed to promote        catch-up growth and support development.        Features:    -   Reduces the need for caloric and vitamin supplementation. More        calories (22 Cal/fl oz) than standard term formulas (20 Cal/fl        oz).    -   Highly absorbed fat blend, with medium-chain triglycerides (MCT        oil) to help meet the special digestive needs of premature        infants.    -   Higher levels of protein, vitamins and minerals per 100 calories        to extend the nutritional support initiated in-hospital.    -   More calcium and phosphorus for improved bone mineralization.        Ingredients: -D Corn syrup solids, nonfat milk, lactose, whey        protein concentrate, soy oil, high-oleic safflower oil,        fractionated coconut oil (medium chain triglycerides), coconut        oil, potassium citrate, calcium phosphate tribasic, calcium        carbonate, ascorbic acid, magnesium chloride, potassium        chloride, sodium chloride, taurine, ferrous sulfate, m-inositol,        choline chloride, ascorbyl palmitate, L-carnitine,        alpha-tocopheryl acetate, zinc sulfate, niacinamide, mixed        tocopherols, sodium citrate, calcium pantothenate, cupric        sulfate, thiamine chloride hydrochloride, vitamin A palmitate,        beta carotene, riboflavin, pyridoxine hydrochloride, folic acid,        manganese sulfate, phylloquinone, biotin, sodium selenite,        vitamin D3 and cyanocobalamin.        G. Similac Natural Care Low-Iron Human Milk Fortifier Ready to        use, 24 Cal/fl oz.:        Usage: Designed to be mixed with human milk or to be fed        alternatively with human milk to low-birth-weight infants.        Ingredients: -D Water, nonfat milk, hydrolyzed cornstarch,        lactose, fractionated coconut oil (medium-chain triglycerides),        whey protein concentrate, soy oil, coconut oil, calcium        phosphate tribasic, potassium citrate, magnesium chloride,        sodium citrate, ascorbic acid, calcium carbonate, mono and        diglycerides, soy lecithin, carrageenan, choline chloride,        m-inositol, taurine, niacinamide, L-carnitine, alpha tocopheryl        acetate, zinc sulfate, potassium chloride, calcium pantothenate,        ferrous sulfate, cupric sulfate, riboflavin, vitamin A        palmitate, thiamine chloride hydrochloride, pyridoxine        hydrochloride, biotin, folic acid, manganese sulfate,        phylloquinone, vitamin D3, sodium selenite and cyanocobalamin.

Various PUFAs of this invention can be substituted and/or added to theinfant formulae described above and to other infant formulae known tothose in the art.

II. Nutritional Formulations

A. ENSURE®

Usage: ENSURE is a low-residue liquid food designed primarily as an oralnutritional supplement to be used with or between meals or, inappropriate amounts, as a meal replacement. ENSURE is lactose- andgluten-free, and is suitable for use in modified diets, includinglow-cholesterol diets. Although it is primarily an oral supplement, itcan be fed by tube.

Patient Conditions:

-   -   For patients on modified diets    -   For elderly patients at nutrition risk    -   For patients with involuntary weight loss    -   For patients recovering from illness or surgery    -   For patients who need a low-residue diet        Ingredients: -D Water, Sugar (Sucrose), Maltodextrin (Corn),        Calcium and Sodium Caseinates, High-Oleic Safflower Oil, Soy        Protein Isolate, Soy Oil, Canola Oil, Potassium Citrate, Calcium        Phosphate Tribasic, Sodium Citrate, Magnesium Chloride,        Magnesium Phosphate Dibasic, Artificial Flavor, Sodium Chloride,        Soy Lecithin, Choline Chloride, Ascorbic Acid, Carrageenan, Zinc        Sulfate, Ferrous Sulfate, Alpha-Tocopheryl Acetate, Gellan Gum,        Niacinamide, Calcium Pantothenate, Manganese Sulfate, Cupric        Sulfate, Vitamin A Palmitate, Thiamine Chloride Hydrochloride,        Pyridoxine Hydrochloride, Riboflavin, Folic Acid, Sodium        Molybdate, Chromium Chloride, Biotin, Potassium Iodide, Sodium        Selenate.        B. ENSURE® BARS:        Usage: ENSURE BARS are complete, balanced nutrition for        supplemental use between or with meals. They provide a        delicious, nutrient-rich alternative to other snacks. ENSURE        BARS contain <1 g lactose/bar, and Chocolate Fudge Brownie        flavor is gluten-free. (Honey Graham Crunch flavor contains        gluten.)        Patient Conditions:    -   For patients who need extra calories, protein, vitamins and        minerals.    -   Especially useful for people who do not take in enough calories        and nutrients.    -   For people who have the ability to chew and swallow    -   Not to be used by anyone with a peanut allergy or any type of        allergy to nuts.        Ingredients: Honey Graham Crunch—High-Fructose Corn Syrup, Soy        Protein Isolate, Brown Sugar, Honey, Maltodextrin (Corn), Crisp        Rice (Milled Rice, Sugar [Sucrose], Salt [Sodium Chloride] and        Malt), Oat Bran, Partially Hydrogenated Cottonseed and Soy Oils,        Soy Polysaccharide, Glycerine, Whey Protein Concentrate,        Polydextrose, Fructose, Calcium Caseinate, Cocoa Powder,        Artificial Flavors, Canola Oil, High-Oleic Safflower Oil, Nonfat        Dry Milk, Whey Powder, Soy Lecithin and Corn Oil. Manufactured        in a facility that processes nuts.        Vitamins and Minerals: Calcium Phosphate Tribasic, Potassium        Phosphate Dibasic, Magnesium Oxide, Salt (Sodium Chloride),        Potassium Chloride, Ascorbic Acid, Ferric Orthophosphate,        Alpha-Tocopheryl Acetate, Niacinamide, Zinc Oxide, Calcium        Pantothenate, Copper Gluconate, Manganese Sulfate, Riboflavin,        Beta Carotene, Pyridoxine Hydrochloride, Thiamine Mononitrate,        Folic Acid, Biotin, Chromium Chloride, Potassium Iodide, Sodium        Selenate, Sodium Molybdate, Phylloquinone, Vitamin D3 and        Cyanocobalamin.        Protein: Honey Graham Crunch—The protein source is a blend of        soy protein isolate and milk proteins.

Soy protein isolate 74% Milk proteins 26%Fat: Honey Graham Crunch—The fat source is a blend of partiallyhydrogenated cottonseed and soybean, canola, high oleic safflower, oils,and soy lecithin.

Partially hydrogenated cottonseed and soybean oil 76%  Canola oil 8%High-oleic safflower oil 8% Corn oil 4% Soy lecithin 4%Carbohydrate: Honey Graham Crunch—The carbohydrate source is acombination of high-fructose corn syrup, brown sugar, maltodextrin,honey, crisp rice, glycerine, soy polysaccharide, and oat bran.

High-fructose corn syrup 24% Brown sugar 21% Maltodextrin 12% Honey 11%Crisp rice  9% Glycerine  9% Soy Polysaccharide  7% Oat bran  7%C. ENSURE® HIGH PROTEIN:Usage: ENSURE HIGH PROTEIN is a concentrated, high-protein liquid fooddesigned for people who require additional calories, protein, vitamins,and minerals in their diets. It can be used as an oral nutritionalsupplement with or between meals or, in appropriate amounts, as a mealreplacement. ENSURE HIGH PROTEIN is lactose- and gluten-free, and issuitable for use by people recovering from general surgery or hipfractures and by patients at risk for pressure ulcers.Patient Conditions:

-   -   For patients who require additional calories, protein, vitamins,        and minerals, such as patients recovering from general surgery        or hip fractures, patients at risk for pressure ulcers, and        patients on low-cholesterol diets        Features:    -   Low in saturated fat    -   Contains 6 g of total fat and <5 mg of cholesterol per serving    -   Rich, creamy taste    -   Excellent source of protein, calcium, and other essential        vitamins and minerals    -   For low-cholesterol diets    -   Lactose-free, easily digested        Ingredients:        Vanilla Supreme: -D Water, Sugar (Sucrose), Maltodextrin (Corn),        Calcium and Sodium Caseinates, High-OIeic Safflower Oil, Soy        Protein Isolate, Soy Oil, Canola Oil, Potassium Citrate, Calcium        Phosphate Tribasic, Sodium Citrate, Magnesium Chloride,        Magnesium Phosphate Dibasic, Artificial Flavor, Sodium Chloride,        Soy Lecithin, Choline Chloride, Ascorbic Acid, Carrageenan, Zinc        Sulfate, Ferrous Suffate, Alpha-Tocopheryl Acetate, Gellan Gum,        Niacinamide, Calcium Pantothenate, Manganese Sulfate, Cupric        Sulfate, Vitamin A Palmitate, Thiamine Chloride Hydrochloride,        Pyridoxine Hydrochloride, Riboflavin, Folic Acid, Sodium        Molybdate, Chromium Chloride, Biotin, Potassium Iodide, Sodium        Selenate, Phylloquinone, Vitamin D3 and Cyanocobalamin.        Protein:

The protein source is a blend of two high-biologic-value proteins:casein and soy.

Sodium and calcium caseinates 85% Soy protein isolate 15%Fat:

The fat source is a blend of three oils: high-oleic safflower, canola,and soy.

High-oleic safflower oil 40% Canola oil 30% Soy oil 30%The level of fat in ENSURE HIGH PROTEIN meets American Heart Association(AHA) guidelines. The 6 grams of fat in ENSURE HIGH PROTEIN represent24% of the total calories, with 2.6% of the fat being from saturatedfatty acids and 7.9% from polyunsaturated fatty acids. These values arewithin the AHA guidelines of <30% of total calories from fat, <10% ofthe calories from saturated fatty acids, and <10% of total calories frompolyunsaturated fatty acids.Carbohydrate:

ENSURE HIGH PROTEIN contains a combination of maltodextrin and sucrose.The mild sweetness and flavor variety (vanilla supreme, chocolate royal,wild berry, and banana), plus VARI-FLAVORS® Flavor Pacs in pecan,cherry, strawberry, lemon, and orange, help to prevent flavor fatigueand aid in patient compliance.

Vanilla and Other Nonchocolate Flavors:

Vanilla and other nonchocolate flavors: Sucrose 60% Maltodextrin 40%Chocolate: Sucrose 70% Maltodextrin 30%D. ENSURE® LIGHTUsage: ENSURE LIGHT is a low-fat liquid food designed for use as an oralnutritional supplement with or between meals. ENSURE LIGHT is lactose-and gluten-free, and is suitable for use in modified diets, includinglow-cholesterol diets.Patient Conditions:

-   -   For normal-weight or overweight patients who need extra        nutrition in a supplement that contains 50% less fat and 20%        fewer calories than ENSURE.    -   For healthy adults who don't eat right and need extra nutrition.        Features:    -   Low in fat and saturated fat    -   Contains 3 g of total fat per serving and <5 mg cholesterol    -   Rich, creamy taste    -   Excellent source of calcium and other essential vitamins and        minerals    -   For low-cholesterol diets    -   Lactose-free, easily digested        Ingredients:        French Vanilla: -D Water, Maltodextrin (Corn), Sugar (Sucrose),        Calcium Caseinate, High-Oleic Safflower Oil, Canola Oil,        Magnesium Chloride, Sodium Citrate, Potassium Citrate, Potassium        Phosphate Dibasic, Magnesium Phosphate Dibasic, Natural and        Artificial Flavor, Calcium Phosphate Tribasic, Cellulose Gel,        Choline Chloride, Soy Lecithin, Carrageenan, Salt (Sodium        Chloride), Ascorbic Acid, Cellulose Gum, Ferrous Sulfate,        Alpha-Tocopheryl Acetate, Zinc Sulfate, Niacinamide, Manganese        Sulfate, Calcium Pantothenate, Cupric Sulfate, Thiamine Chloride        Hydrochloride, Vitamin A Palmitate, Pyridoxine Hydrochloride,        Riboflavin, Chromium Chloride, Folic Acid, Sodium Molybdate,        Biotin, Potassium Iodide, Sodium Selenate, Phylloquinone,        Vitamin D3 and Cyanocobalamin.        Protein:        The protein source is calcium caseinate.

Calcium caseinate 100%Fat:The fat source is a blend of two oils: high-oleic safflower and canola.

High-oleic safflower oil 70% Canola oil 30%The level of fat in ENSURE LIGHT meets American Heart Association (AHA)guidelines. The 3 grams of fat in ENSURE LIGHT represent 13.5% of thetotal calories, with 1.4% of the fat being from saturated fatty acidsand 2.6% from polyunsaturated fatty acids. These values are within theAHA guidelines of <30% of total calories from fat, <10% of the, caloriesfrom saturated fatty acids, and <10% of total calories frompolyunsaturated fatty acids.Carbohydrate:ENSURE LIGHT contains a combination of maltodextrin and sucrose. Thechocolate flavor contains corn syrup as well. The mild sweetness andflavor variety (French vanilla, chocolate supreme, strawberry swirl),plus VARI-FLAVORS® Flavor Pacs in pecan, cherry, strawberry, lemon, andorange, help to prevent flavor fatigue and aid in patient compliance.

Vanilla and other nonchocolate flavors: Sucrose 51% Maltodextrin 49%Chocolate: Sucrose 47.0% Corn Syrup 26.5% Maltodextrin 26.5%Vitamins and Minerals:

An 8-fl-oz serving of ENSURE LIGHT provides at least 25% of the RDIs for24 key vitamins and minerals.

Caffeine:

Chocolate flavor contains 2.1 mg caffeine/8 fl oz.

E. ENSURE PLUS®

Usage: ENSURE PLUS is a high-calorie, low-residue liquid food for usewhen extra calories and nutrients, but a normal concentration ofprotein, are needed. It is designed primarily as an oral nutritionalsupplement to be used with or between meals or, in appropriate amounts,as a meal replacement. ENSURE PLUS is lactose- and gluten-free. Althoughit is primarily an oral nutritional supplement, it can be fed by tube.

Patient Conditions:

-   -   For patients who require extra calories and nutrients, but a        normal concentration of protein, in a limited volume    -   For patients who need to gain or maintain healthy weight        Features:    -   Rich, creamy taste    -   Good source of essential vitamins and minerals        Ingredients:        Vanilla: -D Water, Corn Syrup, Maltodextrin (Corn), Corn Oil,        Sodium and Calcium Caseinates, Sugar (Sucrose), Soy Protein        Isolate, Magnesium Chloride, Potassium Citrate, Calcium        Phosphate Tribasic, Soy Lecithin, Natural and Artificial Flavor,        Sodium Citrate, Potassium Chloride, Choline Chloride, Ascorbic        Acid, Carrageenan, Zinc Sulfate, Ferrous Sulfate,        Alpha-Tocopheryl Acetate, Niacinamide, Calcium Pantothenate,        Manganese Sulfate, Cupric Sulfate, Thiamine Chloride        Hydrochloride, Pyridoxine Hydrochloride, Riboflavin, Vitamin A        Palmitate, Folic Acid, Biotin, Chromium Chloride, Sodium        Molybdate, Potassium Iodide, Sodium Selenite, Phylloquinone,        Cyanocobalamin and Vitamin D3.        Protein:

The protein source is a blend of two high-biologic-value proteins:casein and soy.

Sodium and calcium caseinates 84% Soy protein isolate 16%Fat:

The fat source is corn oil.

Corn oil 100%Carbohydrate:

ENSURE PLUS contains a combination of maltodextrin and sucrose. The mildsweetness and flavor variety (vanilla, chocolate, strawberry, coffee,buffer pecan, and eggnog), plus VARI-FLAVORS® Flavor Pacs in pecan,cherry, strawberry, lemon, and orange, help to prevent flavor fatigueand aid in patient compliance.

Vanilla, Strawberry, Butter Pecan, and Coffee Flavors:

Vanilla, strawberry, butter pecan, and coffee flavors: Corn Syrup 39%Maltodextrin 38% Sucrose 23% Chocolate and eggnog flavors: Corn Syrup36% Maltodextrin 34% Sucrose 30%Vitamins and Minerals:

An 8-fl-oz serving of ENSURE PLUS provides at least 15% of the RDIs for25 key Vitamins and minerals.

Caffeine:

Chocolate flavor contains 3.1 mg Caffeine/8 fl oz. Coffee flavorcontains a trace amount of caffeine.

F. ENSURE PLUS® HN

Usage: ENSURE PLUS HN is a nutritionally complete high-calorie,high-nitrogen liquid food designed for people with higher calorie andprotein needs or limited volume tolerance. It may be used for oralsupplementation or for total nutritional support by tube. ENSURE PLUS HNis lactose- and gluten-free.

Patient Conditions:

-   -   For patients with increased calorie and protein needs, such as        following surgery or injury.    -   For patients with limited volume tolerance and early satiety.        Features:    -   For supplemental or total nutrition    -   For oral or tube feeding    -   1.5 CaVmL,    -   High nitrogen    -   Calorically dense        Ingredients:        Vanilla: -D Water, Maltodextrin (Corn), Sodium and Calcium        Caseinates, Corn Oil, Sugar (Sucrose), Soy Protein Isolate,        Magnesium Chloride, Potassium Citrate, Calcium Phosphate        Tribasic, Soy Lecithin, Natural and Artificial Flavor, Sodium        Citrate, Choline Chloride, Ascorbic Acid, Taurine, L-Carnitine,        Zinc Sulfate, Ferrous Sulfate, Alpha-Tocopheryl Acetate,        Niacinamide, Carrageenan, Calcium Pantothenate, Manganese        Sulfate, Cupric Sulfate, Thiamine Chloride Hydrochloride,        Pyridoxine Hydrochloride, Riboflavin, Vitamin A Palmitate, Folic        Acid, Biotin, Chromium Chloride, Sodium Molybdate, Potassium        Iodide, Sodium Selenite, Phylloquinone, Cyanocobalamin and        Vitamin D3.        G. ENSURE® POWDER:        Usage: ENSURE POWDER (reconstituted with water) is a low-residue        liquid food designed primarily as an oral nutritional supplement        to be used with or between meals. ENSURE POWDER is lactose- and        gluten-free, and is suitable for use in modified diets,        including low-cholesterol diets.        Patient Conditions:    -   For patients on modified diets    -   For elderly patients at nutrition risk    -   For patients recovering from illness/surgery    -   For patients who need a low-residue diet        Features:    -   Convenient, easy to mix    -   Low in saturated fat    -   Contains 9 g of total fat and <5 mg of cholesterol per serving    -   High in vitamins and minerals    -   For low-cholesterol diets    -   Lactose-free, easily digested        Ingredients: -D Corn Syrup, Maltodextrin (Corn), Sugar        (Sucrose), Corn Oil, Sodium and Calcium Caseinates, Soy Protein        Isolate, Artificial Flavor, Potassium Citrate, Magnesium        Chloride, Sodium Citrate, Calcium Phosphate Tribasic, Potassium        Chloride, Soy Lecithin, Ascorbic Acid, Choline Chloride, Zinc        Sulfate, Ferrous Sulfate, Alpha-Tocopheryl Acetate, Niacinamide,        Calcium Pantothenate, Manganese Sulfate, Thiamine Chloride        Hydrochloride, Cupric Sulfate, Pyridoxine Hydrochloride,        Riboflavin, Vitamin A Palmitate, Folic Acid, Biotin, Sodium        Molybdate, Chromium Chloride, Potassium Iodide, Sodium Selenate,        Phylloquinone, Vitamin D3 and Cyanocobalamin.        Protein:

The protein source is a blend of two high-biologic-value proteins:casein and soy.

Sodium and calcium caseinates 84% Soy protein isolate 16%Fat:

The fat source is corn oil.

Corn oil 100%Carbohydrate:

ENSURE POWDER contains a combination of corn syrup, maltodextrin, andsucrose. The mild sweetness of ENSURE POWDER, plus VARI-FLAVORS® FlavorPacs in pecan, cherry, strawberry, lemon, and orange, helps to preventflavor fatigue and aid in patient compliance.

Vanilla: Corn Syrup 35% Maltodextrin 35% Sucrose 30%

H. ENSURE® PUDDING

Usage: ENSURE PUDDING is a nutrient-dense supplement providing balancednutrition in a nonliquid form to be used with or between meals. It isappropriate for consistency-modified diets (e.g., soft, pureed, or fullliquid) or for people with swallowing impairments. ENSURE PUDDING isgluten-free.

Patient Conditions:

-   -   For patients on consistency-modified diets (e.g., soft, pureed,        or full liquid)    -   For patients with swallowing impairments        Features:    -   Rich and creamy, good taste    -   Good source of essential vitamins and minerals    -   Convenient-needs no refrigeration    -   Gluten-free        Nutrient Profile per 5 oz: Calories 250, Protein 10.9%, Total        Fat 34.9%, Carbohydrate 54.2%        Ingredients:        Vanilla: -D Nonfat Milk, Water, Sugar (Sucrose), Partially        Hydrogenated Soybean Oil, Modified Food Starch, Magnesium        Sulfate, Sodium Stearoyl Lactylate, Sodium Phosphate Dibasic,        Artificial Flavor, Ascorbic Acid, Zinc Sulfate, Ferrous Sulfate,        Alpha-Tocopheryl Acetate, Choline Chloride, Niacinamide,        Manganese Sulfate, Calcium Pantothenate, FD&C Yellow #5,        Potassium Citrate, Cupric Sulfate, Vitamin A Palmitate, Thiamine        Chloride Hydrochloride, Pyridoxine Hydrochloride, Riboflavin,        FD&C Yellow #6, Folic Acid, Biotin, Phylloquinone, Vitamin D3        and Cyanocobalamin.        Protein:

The protein source is nonfat milk.

Nonfat milk 100%Fat:

The fat source is hydrogenated soybean oil.

Hydrogenated soybean oil 100%Carbohydrate:

ENSURE PUDDING contains a combination of sucrose and modified foodstarch. The mild sweetness and flavor variety (vanilla, chocolate,butterscotch, and tapioca) help prevent flavor fatigue. The productcontains 9.2 grams of lactose per serving.

Vanilla and other nonchocolate flavors: Sucrose 56% Lactose 27% Modifiedfood starch 17% Chocolate: Sucrose 58% Lactose 26% Modified food starch16%I. ENSURE® WITH FIBER:Usage: ENSURE WITH FIBER is a fiber-containing, nutritionally completeliquid food designed for people who can benefit from increased dietaryfiber and nutrients. ENSURE WITH FIBER is suitable for people who do notrequire a low-residue diet. It can be fed orally or by tube, and can beused as a nutritional supplement to a regular diet or, in appropriateamounts, as a meal replacement. ENSURE WITH FIBER is lactose- andgluten-free, and is suitable for use in modified diets, includinglow-cholesterol diets.Patient Conditions:

-   -   For patients who can benefit from increased dietary fiber and        nutrients        Features:    -   New advanced formula-low in saturated fat, higher in vitamins        and minerals    -   Contains 6 g of total fat and <5 mg of cholesterol per serving    -   Rich, creamy taste    -   Good source of fiber    -   Excellent source of essential vitamins and minerals    -   For low-cholesterol diets    -   Lactose- and gluten-free        Ingredients:        Vanilla: -D Water; Maltodextrin (Corn), Sugar (Sucrose), Sodium        and Calcium Caseinates, Oat Fiber, High-Oleic Safflower Oil,        Canola Oil, Soy Protein Isolate, Corn Oil, Soy Fiber, Calcium        Phosphate Tribasic, Magnesium Chloride, Potassium Citrate,        Cellulose Gel, Soy Lecithin, Potassium Phosphate Dibasic, Sodium        Citrate, Natural and Artificial Flavors, Choline Chloride,        Magnesium Phosphate, Ascorbic Acid, Cellulose Gum, Potassium        Chloride, Carrageenan, Ferrous Sulfate, Alpha-Tocopheryl        Acetate, Zinc Sulfate, Niacinamide, Manganese Sulfate, Calcium        Pantothenate, Cupric Sulfate, Vitamin A Palmitate, Thiamine        Chloride Hydrochloride, Pyridoxine Hydrochloride, Riboflavin,        Folic Acid, Chromium Chloride, Biotin, Sodium Molybdate,        Potassium Iodide, Sodium Selenate, Phylloquinone, Vitamin D3 and        Cyanocobalamin.        Protein:

The protein source is a blend of two high-biologic-value proteins-caseinand soy.

Sodium and calcium caseinates 80% Soy protein isolate 20%Fat:

The fat source is a blend of three oils: high-oleic safflower, canola,and corn.

High-oleic safflower oil 40% Canola oil 40% Corn oil 20%The level of fat in ENSURE WITH FIBER meets American Heart Association(AHA) guidelines. The 6 grams of fat in ENSURE WITH FIBER represent 22%of the total calories, with 2.01% of the fat being from saturated fattyacids and 6.7% from polyunsaturated fatty acids. These values are withinthe AHA guidelines of ≦30% of total calories from fat, <10% of thecalories from saturated fatty acids, and ≦10% of total calories frompolyunsaturated fatty acids.Carbohydrate:

ENSURE WITH FIBER contains a combination of maltodextrin and sucrose.The mild sweetness and flavor variety (vanilla, chocolate, and butterpecan), plus VARI-FLAVORS® Flavor Pacs in pecan, cherry, strawberry,lemon, and orange, help to prevent flavor fatigue and aid in patientcompliance.

Vanilla and other nonchocolate flavors: Maltodextrin 66% Sucrose 25% OatFiber  7% Soy Fiber  2% Chocolate: Maltodextrin 55% Sucrose 36% OatFiber  7% Soy Fiber  2%Fiber:

The fiber blend used in ENSURE WITH FIBER consists of oat fiber and soypolysaccharide. This blend results in approximately 4 grams of totaldietary fiber per 8-fl. oz can. The ratio of insoluble to soluble fiberis 95:5.

The various nutritional supplements described above and known to othersof skill in the art can be substituted and/or supplemented with thePUFAs produced in accordance with the present invention.

J. Oxepa™ Nutritional Product

Oxepa is a low-carbohydrate, calorically dense, enteral nutritionalproduct designed for the dietary management of patients with or at riskfor ARDS. It has a unique combination of ingredients, including apatented oil blend containing eicosapentaenoic acid (EPA from fish oil),γ-linolenic acid (GLA from borage oil), and elevated antioxidant levels.

Caloric Distribution:

Caloric density is high at 1.5 Cal/mL (355 Cal/8 fl oz), to minimize thevolume required to meet energy needs.

The distribution of Calories in Oxepa is shown in Table IV.

TABLE IV Caloric Distribution of Oxepa per 8 fl oz. per liter % of CalCalories 355 1,500 — Fat (g) 22.2 93.7 55.2 Carbohydrate (g) 25 105.528.1 Protein (g) 14.8 62.5 16.7 Water (g) 186 785 —Fat:

-   -   Oxepa contains 22.2 g of fat per 8-fl oz serving (93.7 g/L).    -   The fat source is an oil blend of 31.8% canola oil, 25%        medium-chain triglycerides (MCTs), 20% borage oil, 20% fish oil,        and 3.2% soy lecithin. The typical fatty acid profile of Oxepa        is shown in Table V.    -   Oxepa provides a balanced amount of polyunsaturated,        monounsaturated, and saturated fatty acids, as shown in Table        VI.    -   Medium-chain trigylcerides (MCTs)—25% of the fat blend—aid        gastric emptying because they are absorbed by the intestinal        tract without emulsification by bile acids.

The various fatty acid components of Oxepa™ nutritional product can besubstituted and/or supplemented with the PUFAs produced in accordancewith this invention.

TABLE V Typical Fatty Acid Profile % Total Fatty Acids g/8 fl oz* 9/L*Caproic (6:0) 0.2 0.04 0.18 Caprylic (8:0) 14.69 3.1 13.07 Capric (10:0)11.06 2.33 9.87 Palmitic (16:0) 5.59 1.18 4.98 Palmitoleic 1.82 0.381.62 Stearic 1.94 0.39 1.64 Oleic 24.44 5.16 21.75 Linoleic 16.28 3.4414.49 α-Linolenic 3.47 0.73 3.09 γ-Linolenic 4.82 1.02 4.29Eicosapentaenoic 5.11 1.08 4.55 n-3-Docosapentaenoic 0.55 0.12 0.49Docosahexaenoic 2.27 0.48 2.02 Others 7.55 1.52 6.72Fatty acids equal approximately 95% of total fat.

TABLE VI Fat Profile of Oxepa. % of total calories from fat 55.2Polyunsaturated fatty acids 31.44 g/L Monounsaturated fatty acids 25.53g/L Saturated fatty acids 32.38 g/L n-6 to n-3 ratio 1.75:1 Cholesterol9.49 mg/8 fl oz 40.1 mg/LCarbohydrate:

-   -   The carbohydrate content is 25.0 g per 8-fl-oz serving (105.5        g/L).    -   The carbohydrate sources are 45% maltodextrin (a complex        carbohydrate) and 55% sucrose (a simple sugar), both of which        are readily digested and absorbed.    -   The high-fat and low-carbohydrate content of Oxepa is designed        to minimize carbon dioxide (CO2) production. High CO2 levels can        complicate weaning in ventilator-dependent patients. The low        level of carbohydrate also may be useful for those patients who        have developed stress-induced hyperglycemia.    -   Oxepa is lactose-free.

Dietary carbohydrate, the amino acids from protein, and the glycerolmoiety of fats can be converted to glucose within the body. Throughoutthis process, the carbohydrate requirements of glucose-dependent tissues(such as the central nervous system and red blood cells) are met.However, a diet free of carbohydrates can lead to ketosis, excessivecatabolism of tissue protein, and loss of fluid and electrolytes. Theseeffects can be prevented by daily ingestion of 50 to 100 g of digestiblecarbohydrate, if caloric intake is adequate. The carbohydrate level inOxepa is also sufficient to minimize gluconeogenesis, if energy needsare being met.

Protein:

-   -   Oxepa contains 14.8 g of protein per 8-fl-oz serving (62.5 g/L).    -   The total calorie/nitrogen ratio (150:1) meets the need of        stressed patients.    -   Oxepa provides enough protein to promote anabolism and the        maintenance of lean body mass without precipitating respiratory        problems. High protein intakes are a concern in patients with        respiratory insufficiency. Although protein has little effect on        CO2 production, a high protein diet will increase ventilatory        drive.    -   The protein sources of Oxepa are 86.8% sodium caseinate and        13.2% calcium caseinate.    -   The amino acid profile of the protein system in Oxepa meets or        surpasses the standard for high quality protein set by the        National Academy of Sciences.    -   Oxepa is gluten-free.

1. A plant cell, plant or plant tissue comprising a vector, said vectorcomprising a nucleotide sequence comprising SEQ ID NO:1 operably linkedto a promoter, wherein expression of said nucleotide sequence of saidvector results in production of a polyunsaturated fatty acid by saidplant cell, plant or plant tissue.
 2. The plant cell, plant or planttissue of claim 1 wherein said polyunsaturated fatty acid is arachidonicacid or eicosapentaenoic acid.
 3. A polyunsaturated fatty acid expressedby said plant cell, plant or plant tissue of claim
 1. 4. A plant cell,plant or plant tissue comprising a vector, said vector comprising anucleotide sequence encoding a polypeptide, wherein said polypeptide hasΔ5-desaturase activity and has an amino acid sequence comprising SEQ IDNO:12.